System for detecting infection in synovial fluid

ABSTRACT

The invention provides methods and systems for detecting a biomarker in a synovial fluid wherein the system also includes a control to ensure that the test sample is indeed synovial fluid. The biomarkers and the control for synovial fluid can be identified using proteomic methods, including but not limited to antibody based methods, such as an enzyme-linked immunosorbant assay (ELISA), a radioimmunoassay (RIA), or a lateral flow immunoassay.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 61/590,234, filed Jan. 24, 2012, the contents of which areincorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

Age, arthritis, total joint replacement, diabetes, skin infection andsurgery are some of the predisposing risk factors to joint infection,also known as septic arthritis. While fungus and viruses can becontributors, the most critical source of joint infections is bacterialpathogens due to their rapid growth and destruction of joints. Jointinfection, if not properly diagnosed and treated, can be catastrophic tothe joint and in some cases lead to sepsis and death.

Total joint replacements present a particular challenge. The number oftotal joint replacements in the US is growing dramatically. The babyboomer population is rapidly progressing beyond the age of 50. The needfor total joint replacement is increasing as this population remainsactive and is demanding treatments that will allow them to maintaintheir active lifestyles.

Joint pain is frequently misdiagnosed and is a significant contributorto rising medical costs. The most common causes of joint pain arecrystals (gout, pseudogout), injury, infection, and rheumatoidarthritis. Currently, few tests are available to accurately diagnose thecause of joint pain. In many cases, a blood test is performed, whichfrequently yields vague and ambiguous results (sensitivity less than 80%and specificity less than 70%). Testing the joint fluid at the site ofthe pain is much more accurate because one is evaluating a specificresponse versus a general response.

Diagnostic information obtained via joint fluid analysis has beengenerally considered to be vital in an accurate diagnosis. However,there is no consensus as to which tests are most useful and which shouldbe included in routine analysis. Joint infection is a particularlydifficult problem to diagnose with current technology. Joint infectioncan be catastrophic to the health of the joint and can ultimately leadto sepsis that migrates to the rest of the body.

Thus, there is an urgent need in the art for compositions and methodsfor properly diagnosing joint pain. The present invention addresses thisneed.

SUMMARY OF THE INVENTION

The present invention provides a system for diagnosing joint infectionin a subject, wherein the system detects the presence or absence of abiomarker for joint infection in synovial fluid obtained from thesubject, wherein detection of the presence or absence of the biomarkerdiagnoses joint infection in the subject with at least 90% accuracy.

In one embodiment, the system comprises: a) a first region comprising afirst detection reagent that detects the presence of the biomarker forjoint infection in synovial fluid, wherein the first detector reagentspecifically binds the biomarker, b) a second region comprising aninternal control detector reagent for verification of synovial fluid,wherein the internal control detector reagent specifically binds amarker of synovial fluid; wherein joint infection is diagnosed when thebiomarker and the marker of synovial fluid are detected.

In one embodiment, the joint infection is diagnosed when the marker forsynovial fluid is detected at a higher intensity than the biomarker.

In one embodiment, the biomarker is selected from the group consistingof HNP1-3, ELA-2, BPI, NGAL, Resistin, Thrombospondin, Lactoferrin,IL-1β, IL-8, CRP, TNFα, IL-6, HNE, a2M, VEGF, FGF2, SKALP, IP-10, LMP,Orsomucoid, and any combination thereof.

In one embodiment, the marker of synovial fluid is selected from thegroup consisting of hyaluronic acid (HA), mucopolysaccharide,glucosamine, chondroitin sulfate cartilage oligomeric matrix protein,lumican, lubricin, and any combination thereof.

In one embodiment, the system of the invention detects a desiredbiomarker with a sensitivity and specificity of at least 90% for jointinfection.

In one embodiment, the internal control detector reagent is aggrecan.

The invention also provides a method of diagnosing joint infection in asubject comprising detecting the presence or absence of a biomarker insynovial fluid obtained from a joint in the subject. In one embodiment,the method comprises applying synovial fluid obtained from a joint inthe subject to a system, wherein the system comprises a molecule thatspecifically binds a biomarker for joint infection and a controldetector molecule that specifically binds a marker of synovial fluid,wherein detection of the presence or absence of the biomarker and thedetection of the marker for synovial fluid diagnoses joint infection inthe subject.

In one embodiment, the method comprises a) contacting the synovial fluidobtained from the joint in the subject with an assay buffer, b) applyingthe synovial fluid so contacted to a system comprising: i) a firstregion comprising a first detection reagent that detects the presence ofa biomarker for infection in synovial fluid, wherein the first detectorreagent specifically binds the biomarker, and ii) a second regioncomprising an internal control detector reagent for verification ofsynovial fluid, wherein the internal control detector reagentspecifically binds a marker of synovial fluid; c) diagnosing jointinfection in the patient when the biomarker and marker for synovialfluid are detected.

In one embodiment, joint infection is diagnosed when the second regionis detected at a higher intensity than the first region.

In one embodiment, the biomarker is selected from the group consistingof HNP1-3, ELA-2, BPI, NGAL, Resistin, Thrombospondin, Lactoferrin,IL-1β, IL-8, CRP, TNFα, IL-6, HNE, a2M, VEGF, FGF2, SKALP, IP-10, LMP,Orsomucoid, and any combination thereof.

In one embodiment, the marker of synovial fluid is selected from thegroup consisting of hyaluronic acid (HA), mucopolysaccharide,glucosamine, chondroitin sulfate cartilage oligomeric matrix protein,lumican, lubricin, and any combination thereof.

In one embodiment, the system has a sensitivity and specificity of atleast 90% for joint infection.

In one embodiment, the control detector reagent is aggrecan.

In one embodiment, the assay buffer dilutes the synovial fluid toenhance the ability to pipette and transfer the synovial fluid.

In one embodiment, the assay buffer comprises an agent that lysescellular components present in the synovial fluid.

In one embodiment, the agent is a non-ionic surfactant.

In one embodiment, the assay buffer comprises an agent that preservesthe synovial fluid and stabilizes biomarkers present in the synovialfluid.

In one embodiment, the assay buffer comprises an agent that inhibits aninterfering component present in the synovial fluid.

In one embodiment, the assay buffer maintains a pH in the range of about6-8.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1 is an image of an exemplary system for detecting a biomarker in ajoint fluid. The image shows the basic components of the system andtheir relationship to each other. The system includes bottom or plasticbacking. The system also includes a test line and a control line.

FIG. 2 is a schematic of an exemplary system for detecting a biomarkerin a joint fluid. The image shows the basic components of the system andtheir relationship to each other. The system includes control lineuseful for the detection of hyaluronic acid in order to ensure that thesample tested is indeed synovial fluid.

FIG. 3 is an image of a dot plot summarizing the ROC Analysis for AreaUnder the Curve (AUC) with respect to Interleukin-1α between infectedand aseptic groups.

FIG. 4 is an image of a dot plot summarizing the ROC Analysis for AreaUnder the Curve (AUC) with respect to Interleukin-1β between infectedand aseptic groups.

FIG. 5 is an image of a dot plot summarizing the ROC Analysis for AreaUnder the Curve (AUC) with respect to Interleukin-6 between infected andaseptic groups.

FIG. 6 is an image of a dot plot summarizing the ROC Analysis for AreaUnder the Curve (AUC) with respect to Interleukin-8 between infected andaseptic groups.

FIG. 7 is an image of a dot plot summarizing the ROC Analysis for AreaUnder the Curve (AUC) with respect to Interleukin-10 between infectedand aseptic groups.

FIG. 8 is an image of a dot plot summarizing the ROC Analysis for AreaUnder the Curve (AUC) with respect to Interleukin-17 between infectedand aseptic groups.

FIG. 9 is an image of a dot plot summarizing the ROC Analysis for AreaUnder the Curve (AUC) with respect to G-CSF between infected and asepticgroups.

FIG. 10 is an image of a dot plot summarizing the ROC Analysis for AreaUnder the Curve (AUC) with respect to VEGF between infected and asepticgroups.

FIG. 11 is an image of a dot plot summarizing the ROC Analysis for AreaUnder the Curve (AUC) with respect to SKALP between infected and asepticgroups.

FIG. 12 is an image summarizing the clinical results of HNP1-3. From aclinical standpoint, HNP1-3 showed a clear separation betweenperiprosthetic joint infection positive and negative samples.

FIG. 13 is an image demonstrating that that dilution has a significanteffect on the clinical separation between periprosthetic joint infectionpositive and negative samples.

FIG. 14 is an image of a clinical plot showing that HNP1-3 is abiomarker for periprosthetic joint infection in synovial fluid fromjoint samples.

FIG. 15 is an image showing the results from samples tested includingsynovial fluid from native joints with respect to the presence of HNP1-3in the samples.

FIG. 16 is an image of a clinical plot showing that BPI is a biomarkerfor periprosthetic joint infection in synovial fluid from joint samples.

FIG. 17 is an image demonstrating that BPI is a biomarker forperiprosthetic joint infection in synovial fluid from joint samples.

FIG. 18 is an image showing the measure of HA for serum versus synovialfluid. In the right panel, (−1) denotes serum, (1) denotes synovialfluid.

DETAILED DESCRIPTION

The present invention relates to a system for conveniently detecting thepresence or absence of a biomarker associated with inflammation, as wellas determining variable levels of the biomarker in a sample, preferablya synovial fluid sample.

In one embodiment, the invention provides a system for convenientlydetecting the presence or absence of a biomarker associated withinflammation in a joint. The inflammation can be in a native joint or areplacement joint. In some instances, the inflammation is associatedwith an infection.

In one embodiment, the invention relates to an immunoassay device thatcan be used for detecting a biomarker in a specimen, and an immunoassaymethod using the same.

In another embodiment, the system of the invention may comprise anymethod known in the art to effectively detect a biomarker in a sample.Suitable methods include, but are not limited to, immunoassays, enzymeassays, mass spectrometry, biosensors, and chromatography. Thus, thesystem of the invention includes the use of any type of instrumentalityto detect a desired biomarker.

The invention relates to the discovery that one or more genes andcorresponding polypeptides, wherein the polypeptides have significantamino acid sequence similarity with a family of proteins that includesthe markers of the invention disclosed herein occurs in the synovialfluid of a patient afflicted with infection in a joint of the patient.These polypeptides bind specifically with antibodies that are raisedagainst proteins of that family. Occurrence of these polypeptides in apatient's synovial fluid derived from the infected joint is a diagnosticthat the patient is afflicted with infection in the joint. The amount ofthe polypeptides decreases with effective treatment of the infection ofthe joint. Thus, the polypeptides can also be used to assess theefficacy of any type of therapy directed to the infected joint.

Accordingly, the system of the invention provides a new and convenientplatform for monitoring pathology and response to a particulartreatment. In one embodiment, the system of the invention provides aplatform for detecting a marker of infection in a joint, preferablyperiprosthetic joint infection, with at least 80% sensitivity,preferably at least 90%. In one embodiment, the system of the inventionprovides a platform for detecting a marker of infection in a joint,preferably periprosthetic joint infection with at least 80% specificity,preferably at least 90%. In yet another embodiment, the system allowsfor the detection of the desired marker with at least 80% sensitivity,preferably at least 90% and at least 80% specificity, preferably atleast 90%. In yet another embodiment, the system allows for thedetection of the desired marker with at least 80% accuracy, preferablyat least 90%.

In one embodiment, the system of the invention can be used to diagnosejoint pain, preferably diagnosing the source of inflammation that isassociated with the joint pain. In one embodiment, the system of theinvention can be used to diagnose joint pain associated withinflammation. In some instances, inflammation in the joint can be causedby bacterial infection. In other instances, the inflammation isassociated with periprosthetic joint infection. In one embodiment, jointinfection is diagnosed by detecting the presence of the markers of theinvention in a sample, such as synovial fluid.

In some instances, the system of the invention may take the form of auser-friendly point-of-use or point-of-care platform, for example alateral flow device, having a sample application region and a readabledetection region to indicate the presence or absence of the biomarker orvariable levels of the biomarker. In one embodiment, the readabledetection region includes a test line and a control line, wherein thetest line detects the biomarker associated with the disease or disorder,and the control line detects the presence or absence of a markerassociated with the fluid being tested. Preferably, the fluid beingtested is synovial fluid and the marker for synovial fluid includes, butis not limited to, hyaluronic acid (HA), mucopolysaccharide,glucosamine, chondroitin sulfate cartilage oligomeric matrix protein,lumican, lubricin, and the like. In one embodiment, HA is detected insynovial fluid using an agent that binds to HA. Preferably, the agentthat binds to HA is an anti-HA antibody, more preferably, the agent thatbinds HA is aggrecan.

In one embodiment, a comparison of the control line to the test lineyields the test result from the diagnostic system of the invention. Insome instances, a valid result occurs when the control line is detectedat a higher intensity level than the test line. For example, a validresult occurs when the control line is darker than the test line. Thatis, the control line represents an internal control for the diagnosticsystem of the invention for verifying that the sample being evaluated issynovial fluid.

In one embodiment, the control line is a reference line that insuresthat the test has been run correctly. The control line is also used as areference when the reader determines if the result is positive ornegative. For example, the system of the invention is useful for thediagnosis of infection in a joint when the control line is detected at ahigher intensity than the test line. In some instances, if the test lineis darker than the control line then the test is said to have an invalidresult. If the test line is lighter than the control line then the testis said to have a valid result.

In one embodiment, the system of the invention detects a biomarker byway of a lateral flow immunoassay that utilizes strips of cellulosemembrane onto which antibodies and other reagents are applied. Forexample, the test sample moves along the strip due to capillary actionand reacts with the reagents at different points along the strip. Theend result is the appearance or absence of a detectable line or spot.

In one embodiment, the lateral flow device can be in the form of acartridge that can be read by a machine. Preferably, the machine isautomated.

In one embodiment, the biomarkers of the invention can be detected in asystem that takes the form of a laboratory test, for example a type ofnumbered well plate (e.g., 96 well plate).

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “abnormal” when used in the context of organisms, tissues,cells or components thereof, refers to those organisms, tissues, cellsor components thereof that differ in at least one observable ordetectable characteristic (e.g., age, treatment, time of day, etc.) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics which arenormal or expected for one cell or tissue type, might be abnormal for adifferent cell or tissue type.

As used herein the terms “alteration,” “defect,” “variation,” or“mutation,” refers to a mutation in a gene in a cell that affects thefunction, activity, expression (transcription or translation) orconformation of the polypeptide that it encodes. Mutations encompassedby the present invention can be any mutation of a gene in a cell thatresults in the enhancement or disruption of the function, activity,expression or conformation of the encoded polypeptide, including thecomplete absence of expression of the encoded protein and can include,for example, missense and nonsense mutations, insertions, deletions,frameshifts and premature terminations. Without being so limited,mutations encompassed by the present invention may alter splicing themRNA (splice site mutation) or cause a shift in the reading frame(frameshift).

The term “amplification” refers to the operation by which the number ofcopies of a target nucleotide sequence present in a sample ismultiplied.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)₂, as well as single chain antibodies and humanizedantibodies (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York;Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird etal., 1988, Science 242:423-426).

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. κ and λ light chains refer tothe two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “applicator,” as the term is used herein, is meant anydevice including, but not limited to, a hypodermic syringe, a pipette,an iontophoresis device, a patch, and the like, for administering thecompositions of the invention to a subject.

“Aggregation” means a massing together or clustering of independent butsimilar units, such as particles, parts, or bodies.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequence or a partial nucleotidesequence encoding a protein that elicits an immune response, thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

The term “auto-antigen” means, in accordance with the present invention,any self-antigen which is mistakenly recognized by the immune system asbeing foreign. Auto-antigens comprise, but are not limited to, cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

As used herein, “biomarker” in the context of the present inventionencompasses, without limitation, proteins, nucleic acids, andmetabolites, together with their polymorphisms, mutations, variants,modifications, subunits, fragments, protein-ligand complexes, anddegradation products, protein-ligand complexes, elements, relatedmetabolites, and other analytes or sample-derived measures. Biomarkerscan also include mutated proteins or mutated nucleic acids. Biomarkersalso encompass non-blood borne factors or non-analyte physiologicalmarkers of health status, such as clinical parameters, as well astraditional laboratory risk factors. Biomarkers also include anycalculated indices created mathematically or combinations of any one ormore of the foregoing measurements, including temporal trends anddifferences.

As used herein, a “biosensor” is an analytical device for the detectionof an analyte in a sample. Biosensors can comprise a recognitionelement, which can recognize or capture a specific analyte, and atransducer, which transmits the presence or absence of an analyte into adetectable signal.

As used herein, the term “data” in relation to one or more biomarkers,or the term “biomarker data” generally refers to data reflective of theabsolute and/or relative abundance (level) of a product of a biomarkerin a sample. As used herein, the term “dataset” in relation to one ormore biomarkers refers to a set of data representing levels of each ofone or more biomarker products of a panel of biomarkers in a referencepopulation of subjects. A dataset can be used to generate aformula/classifier of the invention. According to one embodiment, thedataset need not comprise data for each biomarker product of the panelfor each individual of the reference population. For example, the“dataset” when used in the context of a dataset to be applied to aformula can refer to data representing levels of products of eachbiomarker for each individual in one or more reference populations, butas would be understood can also refer to data representing levels ofproducts of each biomarker for 99%, 95%, 90%, 85%, 80%, 75%, 70% or lessof the individuals in each of said one or more reference populations andcan still be useful for purposes of applying to a formula.

The term “control or reference standard” describes a material comprisingnone, or a normal, low, or high level of one of more of the marker (orbiomarker) expression products of one or more the markers (orbiomarkers) of the invention, such that the control or referencestandard may serve as a comparator against which a sample can becompared.

As used herein, a “detector molecule” is a molecule that may be used todetect a compound of interest. Non-limiting examples of a detectormolecule are molecules that bind specifically to a compound of interest,such as, but not limited to, an antibody, a cognate receptor, and asmall molecule.

By the phrase “determining the level of marker (or biomarker)expression” is meant an assessment of the degree of expression of amarker in a sample at the nucleic acid or protein level, usingtechnology available to the skilled artisan to detect a sufficientportion of any marker expression product.

“Differentially increased expression” or “up regulation” refers tobiomarker product levels which are at least 10% or more, for example,20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% higher or more, and/or 1.1fold, 1.2 fold, 1.4 fold, 1.6 fold, 1.8 fold, 2.0 fold higher or more,and any and all whole or partial increments therebetween than a control.

“Differentially decreased expression” or “down regulation” refers tobiomarker product levels which are at least 10% or more, for example,20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% lower or less, and/or 2.0fold, 1.8 fold, 1.6 fold, 1.4 fold, 1.2 fold, 1.1 fold or less lower,and any and all whole or partial increments therebetween than a control.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

As used herein, an “immunoassay” refers to a biochemical test thatmeasures the presence or concentration of a substance in a sample, suchas a biological sample, using the reaction of an antibody to its cognateantigen, for example the specific binding of an antibody to a protein.Both the presence of the antigen or the amount of the antigen presentcan be measured.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of a component of the invention in akit for detecting biomarkers disclosed herein. The instructionalmaterial of the kit of the invention can, for example, be affixed to acontainer which contains the component of the invention or be shippedtogether with a container which contains the component. Alternatively,the instructional material can be shipped separately from the containerwith the intention that the instructional material and the component beused cooperatively by the recipient.

The term “label” when used herein refers to a detectable compound orcomposition that is conjugated directly or indirectly to a probe togenerate a “labeled” probe. The label may be detectable by itself (e.g.radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition that is detectable (e.g., avidin-biotin). Insome instances, primers can be labeled to detect a PCR product.

The “level” of one or more biomarkers means the absolute or relativeamount or concentration of the biomarker in the sample.

A “marker,” as the term is used herein, refers to a molecule that can bedetected. Therefore, a marker according to the present inventionincludes, but is not limited to, a nucleic acid, a polypeptide, acarbohydrate, a lipid, an inorganic molecule, an organic molecule, or aradiolabel, each of which may vary widely in size and properties. A“marker” as used herein can also mean a “biomarker.” A “marker” can bedetected using any means known in the art or by a previously unknownmeans that only becomes apparent upon consideration of the marker by theskilled artisan. A marker may be detected using a direct means, or by amethod including multiple steps of intermediate processing and/ordetection. The term “tag” is also used interchangeably with the term“marker,” but the term “tag” may also be used, in certain aspects, toinclude markers that are associated with one or more other molecules.

The term “marker (or biomarker) expression” as used herein, encompassesthe transcription, translation, post-translation modification, andphenotypic manifestation of a gene, including all aspects of thetransformation of information encoded in a gene into RNA or protein. Byway of non-limiting example, marker expression includes transcriptioninto messenger RNA (mRNA) and translation into protein, as well astranscription into types of RNA such as transfer RNA (tRNA) andribosomal RNA (rRNA) that are not translated into protein.

The terms “microarray” and “array” refers broadly to both “DNAmicroarrays” and “DNA chip(s),” and encompasses all art-recognized solidsupports, and all art-recognized methods for affixing nucleic acidmolecules thereto or for synthesis of nucleic acids thereon. Preferredarrays typically comprise a plurality of different nucleic acid probesthat are coupled to a surface of a substrate in different, knownlocations. These arrays, also described as “microarrays” or colloquially“chips” have been generally described in the art, for example, U.S. Pat.Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195, 5,800,992, 6,040,193,5,424,186 and Fodor et al., 1991, Science, 251:767-777, each of which isincorporated by reference in its entirety for all purposes. Arrays maygenerally be produced using a variety of techniques, such as mechanicalsynthesis methods or light directed synthesis methods that incorporate acombination of photolithographic methods and solid phase synthesismethods. Techniques for the synthesis of these arrays using mechanicalsynthesis methods are described in, e.g., U.S. Pat. Nos. 5,384,261, and6,040,193, which are incorporated herein by reference in their entiretyfor all purposes. Although a planar array surface is preferred, thearray may be fabricated on a surface of virtually any shape or even amultiplicity of surfaces. Arrays may be nucleic acids on beads, gels,polymeric surfaces, fibers such as fiber optics, glass or any otherappropriate substrate. (See U.S. Pat. Nos. 5,770,358, 5,789,162,5,708,153, 6,040,193 and 5,800,992, which are hereby incorporated byreference in their entirety for all purposes.) Arrays may be packaged insuch a manner as to allow for diagnostic use or can be an all-inclusivedevice; e.g., U.S. Pat. Nos. 5,856,174 and 5,922,591 incorporated intheir entirety by reference for all purposes. Arrays are commerciallyavailable from, for example, Affymetrix (Santa Clara, Calif.) andApplied Biosystems (Foster City, Calif.), and are directed to a varietyof purposes, including genotyping, diagnostics, mutation analysis,marker expression, and gene expression monitoring for a variety ofeukaryotic and prokaryotic organisms. The number of probes on a solidsupport may be varied by changing the size of the individual features.In one embodiment the feature size is 20 by 25 microns square, in otherembodiments features may be, for example, 8 by 8, 5 by 5 or 3 by 3microns square, resulting in about 2,600,000, 6,600,000 or 18,000,000individual probe features.

“Measuring” or “measurement,” or alternatively “detecting” or“detection,” means assessing the presence, absence, quantity or amount(which can be an effective amount) of either a given substance within aclinical or subject-derived sample, including the derivation ofqualitative or quantitative concentration levels of such substances, orotherwise evaluating the values or categorization of a subject'sclinical parameters.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

“Polypeptide,” as used herein refers to a polymer in which the monomersare amino acid residues which are joined together through amide bonds.When the amino acids are alpha-amino acids, either the L-optical isomeror the D-optical isomer can be used, the L-isomers being preferred. Theterms “polypeptide” or “protein” or “peptide” as used herein areintended to encompass any amino acid sequence and include modifiedsequences such as glycoproteins. The term “polypeptide” or “protein” or“peptide” is specifically intended to cover naturally occurringproteins, as well as those which are recombinantly or syntheticallyproduced. It should be noted that the term “polypeptide” or “protein”includes naturally occurring modified forms of the proteins, such asglycosylated forms.

A “reference level” of a biomarker means a level of the biomarker thatis indicative of a particular disease state, phenotype, or lack thereof,as well as combinations of disease states, phenotypes, or lack thereof.A “positive” reference level of a biomarker means a level that isindicative of a particular disease state or phenotype. A “negative”reference level of a biomarker means a level that is indicative of alack of a particular disease state or phenotype.

“Sample” or “biological sample” as used herein means a biologicalmaterial isolated from an individual. The biological sample may containany biological material suitable for detecting the desired biomarkers,and may comprise cellular and/or non-cellular material obtained from theindividual.

The term “solid support,” “support,” and “substrate” as used herein areused interchangeably and refer to a material or group of materialshaving a rigid or semi-rigid surface or surfaces. In one embodiment, atleast one surface of the solid support will be substantially flat,although in some embodiments it may be desirable to physically separatesynthesis regions for different compounds with, for example, wells,raised regions, pins, etched trenches, or the like. According to otherembodiments, the solid support(s) will take the form of beads, resins,gels, microspheres, or other geometric configurations. See U.S. Pat. No.5,744,305 for exemplary substrates.

As used herein, the term “wild-type” refers to a gene or gene productisolated from a naturally occurring source. A wild-type gene is thatwhich is most frequently observed in a population and is thusarbitrarily designed the “normal” or “wild-type” form of the gene. Incontrast, the term “modified” or “mutant” refers to a gene or geneproduct that displays modifications in sequence and/or functionalproperties (i.e., altered characteristics) when compared to thewild-type gene or gene product. It is noted that naturally occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics (including altered nucleic acid sequences) whencompared to the wild-type gene or gene product.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

The present invention relates to systems and methods for convenientlymonitoring the presence or absence of a biomarker in a joint fluid, aswell as determining variable levels of the biomarker joint fluid.Preferably, the joint fluid is synovial fluid.

The invention provides methods and systems for detecting a biomarker ina synovial fluid wherein the system also includes a control in order toensure that the test sample is indeed synovial fluid. The biomarkers andthe control for synovial fluid may be identified by any suitable assay.A suitable assay may include one or more of an enzyme assay, animmunoassay, mass spectrometry, chromatography, electrophoresis, abiosensor, an antibody microarray, or any combination thereof. If animmunoassay is used it may be an enzyme-linked immunosorbant immunoassay(ELISA), a sandwich assay, a competitive assay, a radioimmunoassay(RIA), a lateral flow immunoassay, a Western Blot, an immunoassay usinga biosensor, an immunoprecipitation assay, an agglutination assay, aturbidity assay or a nephelometric assay.

Accordingly, the invention includes any platform for detecting a desiredbiomarker in a biological sample such as synovial fluid. In oneembodiment, the system provides a convenient point-of-care device whichcan quickly detect the presence or absence of a biomarker in an at homeor clinical setting. One non-limiting example of a point of care deviceis a lateral flow immunoassay, which utilizes strips of a membrane,preferably a cellulose membrane, onto which antibodies and otherreagents are applied. The sample moves along the strip due to capillaryaction and reacts with the reagents at different points along the strip.The end result is the appearance or absence of a colored line or spot,which can be compared to a control line. In some instances, the controlline is useful for the detection of a marker of synovial fluid (e.g.,hyaluronic acid) in order to ensure that the sample tested is indeedsynovial fluid. Preferably, the marker of synovial fluid is present at aconcentration significantly different in synovial fluid compared to theamount in other common matrices (i.e. blood) so as to validate that thesample tested is synovial fluid.

In one embodiment, the system may include a base or support layer and anabsorbent matrix comprising at least one absorbent layer through which aliquid sample can flow along a flow path by force or by capillaryaction. The base layer may also be absorbent and be in fluidcommunication with the absorbent matrix, such that the flow path ofliquid sample passes through both the absorbent matrix and the baselayer. The flow path includes at least two regions, where the firstregion is a sample application region, and the second region is adetection region.

In one embodiment, the biomarkers of the invention can be detected in asystem that takes the form of a laboratory test, for example a type ofnumbered well plate (e.g., 96 well plate). In one embodiment, thelateral flow device can be in the form of a cartridge that can be readby a machine. Preferably, the machine is automated.

Processing of Synovial Fluid

Synovial fluid is a biological fluid that is found in the synovialcavity of the joints (e.g., knee, hip, shoulder) of the human bodybetween the cartilage and synovium of facing articulating surfaces.Synovial fluid provides nourishment to the cartilage and also serves asa lubricant for the joints. The cells of the cartilage and synoviumsecrete fluid and the fluid lubricates and reduces friction between thearticulating surfaces.

Human synovial fluid is comprised of approximately 85% water. It isderived from the dialysate of blood plasma, which itself is made up ofwater, dissolved proteins, glucose, clotting factors, mineral ions,hormones, etc. The proteins, albumin and globulins, are present insynovial fluid and are believed to play an important role in thelubrication of the joint area. Other proteins are also found in humansynovial fluid, including the glycoproteins such as alpha-1-acidglycoprotein (AGP), alpha-1-antitrypsin (A1AT) and lubricin.

Another compound that is present in human synovial fluid is hyaluronicacid. Hyaluronic acid is also believed to play a role in lubrication.Human synovial fluid further includes other compounds, such aspolysaccharides and phospholipids. The phospholipid,dipalmitoylphosphatidylcholine (DPPC), is also present in human synovialfluid. DPPC is generally regarded as surfactant and is also believed toplay a role in the lubrication of the joint.

Synovial fluid can be withdrawn from a desired joint for use in thediagnostic system of the invention. The synovial fluid withdrawn can beanalyzed in order to obtain clues for the local condition and to receiveinformation about the disease present in the joint. Physical andchemical properties, inflammation markers, presence of leukocytes,antibodies, and the likes can be investigated to diagnose infection inthe joint.

Accordingly, the invention provides compositions and methods fordetecting the presence of an infection marker in synovial fluid for thediagnosis of the type of infection in a joint as well as a controlmarker that is associated with the synovial fluid being evaluated.Preferably, control marker for synovial fluid includes, but is notlimited to, hyaluronic acid (HA), mucopolysaccharide, glucosamine,chondroitin sulfate cartilage oligomeric matrix protein, lumican,lubricin, and the like.

In one embodiment, the concentration of these synovial fluid markers canbe used as a normalizing factor in the systems and assays of the presentinvention.

Synovial fluid is inherently viscous and presents significant issueswhen the sample is aspirated or pipetted. Without wishing to be bound byany particular theory, the ideal diluent for synovial fluid contains abuffer capable of maintaining a pH in the range of 6-8. Preferably, thebuffer contains saline as a base (i.e. phosphate, Tris). In oneembodiment, the buffer contains a detergent that is capable of lysingthe cellular material in the synovial fluid sample.

Detergents are amphipathic molecules, meaning they contain both anonpolar “tail” having aliphatic or aromatic character and a polar“head.” Ionic character of the polar head group forms the basis forbroad classification of detergents; they may be ionic (charged, eitheranionic or cationic), nonionic (uncharged) or zwitterionic (having bothpositively and negatively charged groups but with a net charge of zero).Detergent molecules allow the dispersion (miscibility) ofwater-insoluble, hydrophobic compounds into aqueous media, including theextraction and solubilization of membrane proteins.

In one embodiment, the buffer of the invention comprises one or morenon-ionic detergents, including, but not limited to,N-octyl-β-D-glucopyranside, N-octyl-β-D-maltoside, ZWITTERGENT 3.14,deoxycholate; n-Dodecanoylsucrose; n-Dodecyl-β-D-glucopyranoside;n-Dodecyl-β-D-maltoside; n-Octyl-β-D-glucopyranoside;n-Octyl-β-D-maltopyranoside; n-Octyl-β-D-thioglucopyranoside;n-Decanoylsucrose; n-Decyl-β-D-maltopyranoside;n-Decyl-β-D-thiomaltoside; n-Heptyl-β-D-glucopyranoside;n-Heptyl-β-D-thioglucopyranoside; n-Hexyl-β-D-glucopyranoside;n-Nonyl-β-D-glucopyranoside; n-Octanoylsucrose;n-Octyl-β-D-glucopyranoside; n-Undecyl-β-D-maltoside; APO-10; APO-12;Big CHAP; Big CHAP, Deoxy; BRIJ® 35; C₁₂E₅; C₁₂E₆; C₁₂E₈; C₁₂E₉;Cyclohexyl-n-ethyl-β-D-maltoside; Cyclohexyl-n-hexyl-β-D-maltoside;Cyclohexyl-n-methyl-β-D-maltoside; Digitonin; ELUGENT™; GENAPOL® C-100;GENAPOL® X-080; GENAPOL® X-100; HECAMEG; MEGA-10; MEGA-8; MEGA-9; NOGA;NP-40; PLURONIC® F-127; TRITON® X-100; TRITON® X-114; TWEEN® 20; orTWEEN® 80. Additionally, an ionic detergent can be used with the methodsof the invention, including, but not limited to BATC,Cetyltrimethylammonium Bromide, Chenodeoxycholic Acid, Cholic Acid,Deoxycholic Acid, Glycocholic Acid, Glycodeoxycholic Acid,Glycolithocholic Acid, Lauroylsarcosine, Taurochenodeoxycholic Acid,Taurocholic Acid, Taurodehydrocholic Acid, Taurolithocholic Acid,Tauroursodeoxycholic Acid, and TOPPA. Zwitterionic detergents can alsobe used with the compositions and methods of the invention, including,but not limited to, amidosulfobetaines, CHAPS, CHAPSO, carboxybetaines,and methylbetaines. Anionic detergents can also be used with thecompositions and methods of the invention, including, but not limitedto, e.g. SDS, N-lauryl sarcosine, sodium deoxycholate, alkyl-arylsulphonates, long chain (fatty) alcohol sulphates, olefine sulphates andsulphonates, alpha olefine sulphates and sulphonates, sulphatedmonoglycerides, sulphated ethers, sulphosuccinates, alkane sulphonates,phosphate esters, alkyl isethionates, and sucrose esters.

Generally any suitable liquid may be used as a solvent in the buffer ofthe present invention. The liquid may be organic or inorganic and may bea pure liquid, a mixture of liquids or a solution of substances in theliquid and may contain additional substances to enhance the propertiesof the solvent. Any liquid that is suitable for solubilizing thecellular components of body samples in total or in parts may be regardedas a lysis buffer as used herein.

In one embodiment, the solvent is designed, so that cells, cell debris,nucleic acids, polypeptides, lipids and other biomolecules potentiallypresent in the sample are dissolved. In further embodiments of thepresent invention, the solvent may be designed to assure differentiallysis of specific components of the body sample, leaving othercomponents undissolved.

In some instances, the lysis buffer of the invention comprises one ormore agents that prevent the degradation of components within thesample. Such components may for example comprise enzyme inhibitors suchas proteinase inhibitors, RNAse inhibitors, DNAse inhibitors, nuclease(e.g. endonucleases and exonucleases) inhibitors, etc. Proteinaseinhibitors may e.g. comprise inhibitors of serine proteinases,inhibitors of cysteine proteinases, inhibitors of aspartic proteinases,inhibitors of acidic proteinases, inhibitors of alkaline proteinases orinhibitors of neutral proteinases.

In one embodiment, the ideal diluent for processing synovial fluidcontains a buffer capable of maintaining a pH in the range of about 5 toabout 9, preferably about 6 to about 8, more preferably about 6.5 toabout 7.5. Suitable, but non-limiting, buffers include HEPES, PIPES,Tris-Hydrochloride (Tris-HCl), and MOPS.

Optional components for the diluent may be included as part of thecomposition or as an adjuvant to be added separately, depending on whatsubsequent purification procedures are performed. Optional componentsinclude a defoaming agent at a concentration of about 1%; enzymes suchas lysozyme, lyticase, zymolyase, neuraminidase, streptolysin,cellulysin, mutanolysin, chitinase, glucalase or lysostaphin may beused, at a concentration of about 0.1 to 5 mg/ml; one or more inorganicsalts such as sodium chloride, potassium chloride, magnesium chloride,calcium chloride, lithium chloride, or praseodymium chloride at aconcentration of about 1 mM to 5M; protease inhibitors (e.g.,phenylmethylsulfonyl fluoride, trypsin inhibitor, aprotinin, pepstatinA), reducing reagents (e.g., 2-mercaptoethanol and dithiothreitil) atconcentrations of 0.1 to 10 mM; chelating agents (e.g., disodiumethylenediaminetetraacetic acid (Na₂EDTA), EGTA, CDTA, most preferablyat a concentration of about 1 mM or less); one or more ribonucleases(RNase A, T1, T2, and the like) at concentrations ranging from 1 to 400ug/ml, or any combination of the foregoing. DNase I concentrations mayrange from 1 to 100 units (10,000 units/mg). Preservatives such asProclin 950 can be added to the diluent in order to preserve thesolution comprising synovial fluid from degradation.

The diluent may also include the addition of heterophilic and Rf factorblocking agents to remove the impact of anti-species antibodies and Rffactor that may exist in the clinical sample. Reagents and methods ofthe present disclosure generally inhibit interferents from interferingwith analysis for a particular analyte. Therefore, it is desirable tosubstantially suppress a false positive or a false negative signalcaused by an interferent, if present, in a sample. In one aspect, suchinterferents may be, e.g., a heterophilic antibody, a rheumatoid factor,a lipoprotein, a fibrin, a clotting factor, an IgE, a human antibody toallergens, a human anti-mouse immunoglobulin, a human anti-goatimmunoglobulin, a human anti-bovine immunoglobulin, a human anti-dogimmunoglobulin and a human anti-rabbit immunoglobulin, etc.

Generally, interfering factors (interferents) such as heterophilicantibodies can arise from iatrogenic and noniatrogenic causes. Theformer may result from the normal response of the human immune system toan administered “foreign” protein antigen. The use of diagnostic orpharmaceutical reagents may lead to the introduction of such proteinsand subsequent generation of such antibodies. For example, mousemonoclonal antibodies are foreign proteins in humans and in vivo theymay trigger an immune response to produce human anti-mouse antibodies.In many circumstances where mouse monoclonal antibodies have beenadministered to subjects, those subjects have developed human anti-mouseantibody response.

Accordingly, it is desirable to process synovial fluid and to arrive atan assay buffer that: 1) dilutes the synovial fluid sample to enhancethe ability to pipette/transfer the sample, 2) lyses all of the cellularcomponents in the synovial fluid sample, 3) preserves the synovial fluidsample and stabilizes the biomarkers therefrom, and 4) complexes/removesinterfering substances from the synovial fluid sample.

In some instances, it is desirable to centrifuge (e.g., spin) thesynovial fluid sample prior to assaying the sample. For example, ifthere is some contamination of the synovial fluid with blood, it isdesirable to spin the sample prior to processing in the assay. Howeverin other instances, spinning the synovial fluid is an option whether ornot the synovial fluid is contaminated. This is because the resultspresented herein demonstrate that the differences in the measured levelof a biomarker in some instances is not great enough to impact clinicaldecisions such as where the cutoff is set or whether or not a sample isdeemed to be positive or negative for an infection.

Identifying a Marker or Biomarker

The invention includes methods for the identification of differentiallyexpressed markers between samples of non-infected joint and infectedjoint, as well as methods for the detection of the expression productsof differentially expressed markers of non-infected joint and infectedjoint. In one embodiment, the joint can be a native joint (e.g., RA,Gout, Pseudogout) or a replacement joint. In one embodiment, theinvention provides a method for detecting periprosthetic infection, alsoknown as “septic failure.”

The invention contemplates the identification of differentiallyexpressed markers by whole genome nucleic acid microarray, to identifymarkers differentially expressed between non-infected joint and infectedjoint. The invention further contemplates using methods known to thoseskilled in the art to detect and to measure the level of differentiallyexpressed marker expression products, such as RNA and protein, tomeasure the level of one or more differentially expressed markerexpression products.

In one embodiment, the invention includes a gene signature differentialanalysis method designed to detect genes present in one sample set, andabsent in another. Genes with differential expression in cells fromsites of infection or inflammation versus normal tissue are betterdiagnostic and therapeutic targets than genes that do not change inexpression.

Analysis for the purpose of monitoring differential gene expression maybe focused on a variety of tissues and fluids, and may also be used todetect or measure a number of different molecular targets. When a cellexpresses a gene, it transcribes the appropriate RNA, which isultimately translated into a protein. The relevant protein may then belocalized to a variety of intracellular or extracellular locations.

Methods of detecting or measuring gene expression may utilize methodsthat focus on cellular components (cellular examination), or methodsthat focus on examining extracellular components (fluid examination).Because gene expression involves the ordered production of a number ofdifferent molecules, a cellular or fluid examination may be used todetect or measure a variety of molecules including RNA, protein, and anumber of molecules that may be modified as a result of the protein'sfunction. Typical diagnostic methods focusing on nucleic acids includeamplification techniques such as PCR and RT-PCR (including quantitativevariants), and hybridization techniques such as in situ hybridization,microarrays, blots, and others. Typical diagnostic methods focusing onproteins include binding techniques such as ELISA, immunohistochemistry,microarray and functional techniques such as enzymatic assays.

The practice of the present invention may also employ software andsystems. Computer software products of the invention typically includecomputer readable medium having computer-executable instructions forperforming the logic steps of the method of the invention. Suitablecomputer readable medium include floppy disk, CD-ROM/DVD/DVD-ROM,hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc. Thecomputer executable instructions may be written in a suitable computerlanguage or combination of several languages. Basic computationalbiology methods are described in, for example Setubal and Meidanis etal., Introduction to Computational Biology Methods (PWS PublishingCompany, Boston, 1997); Salzberg, Searles, Kasif, (Ed.), ComputationalMethods in Molecular Biology, (Elsevier, Amsterdam, 1998); Rashidi andBuehler, Bioinformatics Basics: Application in Biological Science andMedicine (CRC Press, London, 2000) and Ouelette and BzevanisBioinformatics: A Practical Guide for Analysis of Gene and Proteins(Wiley & Sons, Inc., 2nd ed., 2001). See U.S. Pat. No. 6,420,108.

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, U.S. Pat.Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555,6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170. Additionally,the present invention may have preferred embodiments that includemethods for providing genetic information over networks such as theInternet as shown in US Pub No 20020183936.

The genes identified as being differentially expressed may be assessedin a variety of nucleic acid detection assays to detect or quantify theexpression level of a gene or multiple genes in a given sample. Forexample, traditional Northern blotting, nuclease protection, RT-PCR,microarray, and differential display methods may be used for detectinggene expression levels. Methods for assaying for mRNA include Northernblots, slot blots, dot blots, and hybridization to an ordered array ofoligonucleotides. Any method for specifically and quantitativelymeasuring a specific protein or mRNA or DNA product can be used.However, methods and assays are most efficiently designed with array orchip hybridization-based methods for detecting the expression of a largenumber of genes. Any hybridization assay format may be used, includingsolution-based and solid support-based assay formats.

The protein products of the genes identified herein can also be assayedto determine the amount of expression. Methods for assaying for aprotein include Western blot, immunoprecipitation, and radioimmunoassay.The proteins analyzed may be localized intracellularly (most commonly anapplication of immunohistochemistry) or extracellularly (most commonlyan application of immunoassays such as ELISA).

Biological samples may be of any biological tissue or fluid containingleukocytes. Frequently the sample will be a “clinical sample” which is asample derived from a patient. Typical clinical samples include, but arenot limited to, synovial fluid, sputum, blood, blood-cells (e.g., whitecells), tissue or fine needle biopsy samples, urine, peritoneal fluid,cerebrospinal fluid, abscesses, and pleural fluid, or cells therefrom.Biological samples may also include sections of tissues, such as frozensections or formalin fixed sections taken for histological purposes.Periprosthetic tissues are often analyzed for evidence of infection.

Controls groups may either be normal (i.e., not infected or not-inflamedtissue, for example) or samples from known types or stages of infectionor inflammation or other disease. As described below, comparison of theexpression patterns of the sample to be tested with those of thecontrols can be used to diagnose infection in the joint. In someinstances, the control groups are only for the purposes of establishinginitial cutoffs for the assays of the invention. Therefore, in someinstances, the systems and methods of the invention can diagnoseinfection in a joint without the need to compare with a control group.

Biomarkers

In one embodiment, the system disclosed herein includes application ofsynovial fluid obtained from a test sample to a system for the detectionof one or more biomarkers that are upregulated in inflammation in ajoint, preferably by at least two-fold increase compared to a normaljoint. In some instances, the inflammation is associated with aninfection (e.g., joint infection). Joint infection can be in a nativejoint or a replacement joint. Preferably, the joint infection is aperiprosthetic joint infection. Such biomarkers include, but are notlimited to, IL-1a (Interleukin 1-alpha), IL-1β (Interleukin 1-beta),IL-1ra (Interleukin 1 receptor antagonist), IL-4 (Interleukin 4), IL-5(Interleukin 5), IL-6 (Interleukin 6), IL-8 (Interleukin 8), IL-10(Interleukin 10), IL-17 (Interleukin 17), ENA-78 (Epithelialcell-derived neutrophil-activating peptide 78), FGF-Basic (Fibroblastgrowth factor basic), G-CSF (Granulocyte colony-stimulating factor),GM-CSF (Granulocyte monocyte colony-stimulating factor), IFN-g(Interferon gamma), MCP-1 (Monocyte chemotactic protein 1), MIP-1a(Macrophage inflammatory protein 1-alpha), MIP-1B (Macrophageinflammatory protein 1-beta), Rantes (Regulated upon Activation, NormalT-cell Expressed, and Secreted), TNF-a (Tumor necrosis factor alpha),Tpo (Thrombopoietin), VEGF (Vascular endothelial growth factor), SKALP(Skin derived antileukoproteinase), SLP-1 (Secretory leukocyte peptidaseinhibitor), CRP (C-Reactive Protein), a-2M (Alpha-2-macroglobulin), LE(Leucocyte Esterase), PCT (Procalcitonin), LBP (Liposaccaride bindingprotein), CGRP (Calictonin gene-related peptide), and the like.Exemplary biomarkers that are upregulated in septic as compared toaseptic inflammation include those listed in U.S. Pat. No. 7,598,080,which is incorporated by reference herein in its entirety. Septicinflammation in the joint can be caused by an infection of viral,bacterial, or parasitic origin.

In one embodiment, the system disclosed herein includes application of asynovial fluid from a test sample to a system for the detection of oneor more biomarker that is upregulated in joint infection. The jointinfection can be in a native joint or a replacement joint. Preferably,the joint infection is a periprosthetic joint infection. Such biomarkersinclude, but are not limited to, IL-1β, IL-6, IL-8, TNFα, G-CSF, IL-1a,VEGF, IP-10, BFGF (aka FGF2), CRP, a2M, SKALP, HNE Enzyme assay,Lactoferrin, Lipocalin-2/NGAL, Neutrophil Elastase-2 (ELA2), Resistin,Thrombospondin-1 (TSP-1), HNP1-3, and BPI.

In another embodiment, the system disclosed herein includes applicationof synovial fluid obtained from a test sample to system for thedetection of one or more biomarkers that are down regulated in jointinfection, preferably by at least two-fold increase compared to a normaljoint. The joint infection can be in a native joint or a replacementjoint. Preferably, the joint infection is a periprosthetic jointinfection. Exemplary biomarkers that are down regulated in septic ascompared to aseptic inflammation include those listed in U.S. Pat. No.7,598,080. In some instances, biomarkers that are downregulated inseptic inflammation include biomarkers that are upregulated in gout.Such biomarkers include, but are not limited to, fatty acid bindingprotein 5 (psoriasis-associated), CD36 antigen (collagen type Ireceptor, thrombospondin receptor), CD9 antigen (p24), lipase A(lysosomal acid, cholesterol esterase (Wolman disease)), glycoprotein(transmembrane) nmb, Fc fragment of IgE (high affinity I, receptor for;alpha polypeptide), potassium channel tetramerisation domain containing12, membrane-spanning 4-domains (subfamily A, member 4), legumain,fibronectin 1, V-set and immunoglobulin domain containing 4, v-mafmusculoaponeurotic fibrosarcoma oncogene homolog B (avian), chondroitinsulfate proteoglycan 2 (versican), histamine N-methyltransferase,disabled homolog 2, mitogen-responsive phosphoprotein (Drosophila),cytochrome P450 (family 1, subfamily B, polypeptide 1),carboxypeptidase, vitellogenic-like, serine dehydratase, high mobilitygroup nucleosomal binding domain 3, annexin A4, and the like.

The present invention is partly based on the discovery that the cells inan inflamed knee, despite appearing the same irrespective of the sourceof the inflammation, have different and diagnostic gene expressionprofiles. For example, a diagnostic gene expression signature andcorresponding protein expression signature can be obtained by comparingresults in cells present in synovial fluid from patients with confirmedbacterial infection as compared to patients with aseptic loosening orpatients with inflammation that is not caused by infection (e.g., gout).Controls may include normal synovial fluid (i.e., not infected ornot-inflamed synovial fluid, for example) or synovial fluid obtainedfrom joints having known types or stages of infection or inflammation.As described elsewhere herein, comparison of the expression patterns ofthe sample to be tested with those of the controls is used to establishinitial cutoffs for the systems of the invention.

The system of the invention can be used to detect at least one, two,three, four, five, or at least ten different bio markers. In someexamples, the system includes determining a proteomic profile. In otherexamples, the system of the invention includes detecting a proteomicprofile including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all ofthese proteins, including any of the proteins set forth in herein orthose listed in U.S. Pat. No. 7,598,080. In one embodiment of theinvention, the system can detect nucleic acids that encode the proteinbiomarker or biomarkers of the invention.

In one embodiment, the invention provides a system for detecting abiomarker of infection in a joint, preferably periprosthetic jointinfection, with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 100% sensitivity; at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 100% specificity; or both at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 100% sensitivity and specificity. In oneembodiment, the invention provides a system for detecting a biomarker ofinfection in joint with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 100% accuracy.

In one embodiment, the system is able to detect HNP1-3 as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity. The cut-off value (derived by ROC analysis) for HNP1-3 as amarker is about 5000 ng/ml, preferably about 6000 ng/ml, preferablyabout 7000 ng/ml, preferably about 8000 ng/ml, preferably about 9000ng/ml, preferably about 10000 ng/ml, most preferably about 7720 ng/ml.The cut-off range for HNP1-3 as a marker for periprosthetic jointinfection with at least 90% sensitivity and specificity is about 1000ng/ml-19000 ng/ml, preferably about 2000 ng/ml-16000 ng/ml, preferablyabout 3000 ng/ml-13000 ng/ml, most preferably about 3334 ng/ml-10946ng/ml.

In one embodiment, the system is able to detect ELA-2 as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity. The cut-off value (derived by ROC analysis) for ELA-2 as amarker is about 600 ng/ml, preferably about 700 ng/ml, preferably about800 ng/ml, preferably about 900 ng/ml, preferably about 1000 ng/ml,preferably about 1100 ng/ml, preferably about 1200 ng/ml, mostpreferably about 942 ng/ml. The cut-off range for ELA-2 as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity is about 400 ng/ml-28000 ng/ml, preferably about 500ng/ml-25000 ng/ml, preferably about 600 ng/ml-22000 ng/ml, mostpreferably about 721 ng/ml-19000 ng/ml.

In one embodiment, the system is able to detect NGAL as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity. The cut-off value (derived by ROC analysis) for NGAL as amarker is about 1400 ng/ml, preferably about 1500 ng/ml, preferablyabout 1600 ng/ml, preferably about 1700 ng/ml, preferably about 1800ng/ml, preferably about 1900 ng/ml, most preferably about 1644 ng/ml.The cut-off range for NGAL as a marker for periprosthetic jointinfection with at least 90% sensitivity and specificity is about 800ng/ml-3600 ng/ml, preferably about 900 ng/ml-3500 ng/ml, preferablyabout 1000 ng/ml-3400 ng/ml, most preferably about 1100 ng/ml-3200ng/ml.

In one embodiment, the system is able to detect Resistin as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity. The cut-off value (derived by ROC analysis) for Resistin asa marker is about 50 ng/ml, preferably about 60 ng/ml, preferably about70 ng/ml, preferably about 80 ng/ml, preferably about 90 ng/ml,preferably about 100 ng/ml, preferably about 110 ng/ml, most preferablyabout 82.9 ng/ml. The cut-off range for Resistin as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity is about 30 ng/ml-150 ng/ml, preferably about 40 ng/ml-140ng/ml, preferably about 50 ng/ml-130 ng/ml, most preferably about 53ng/ml-112 ng/ml.

In one embodiment, the system is able to detect Thrombospondin as amarker for periprosthetic joint infection with at least 90% sensitivityand specificity. The cut-off value (derived by ROC analysis) forThrombospondin as a marker is about 100 ng/ml, preferably about 110ng/ml, preferably about 120 ng/ml, preferably about 130 ng/ml,preferably about 140 ng/ml, preferably about 150 ng/ml, preferably about160 ng/ml, most preferably about 136 ng/ml. The cut-off range forThrombospondin as a marker for periprosthetic joint infection with atleast 90% sensitivity and specificity is about 100 ng/ml-170 ng/ml,preferably about 110 ng/ml-160 ng/ml, preferably about 120 ng/ml-150ng/ml, most preferably about 131 ng/ml-141 ng/ml.

In one embodiment, the system is able to detect Lactoferrin as a markerfor periprosthetic joint infection with at least 90% sensitivity andspecificity. The cut-off value (derived by ROC analysis) for Lactoferrinas a marker is about 2700 ng/ml, preferably about 2800 ng/ml, preferablyabout 2900 ng/ml, preferably about 3000 ng/ml, preferably about 3100ng/ml, preferably about 3200 ng/ml, preferably about 3300 ng/ml, mostpreferably about 2993 ng/ml. The cut-off range for Lactoferrin as amarker for periprosthetic joint infection with at least 90% sensitivityand specificity is about 900 ng/ml-5000 ng/ml, preferably about 1000ng/ml-4900 ng/ml, preferably about 1100 ng/ml-4800 ng/ml, mostpreferably about 1200 ng/ml-4700 ng/ml.

In one embodiment, the system is able to detect IL-113 as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity. The cut-off value (derived by ROC analysis) for IL-1β as amarker is about 20 pg/ml, preferably about 25 pg/ml, preferably about 30pg/ml, preferably about 35 pg/ml, preferably about 40 pg/ml, preferablyabout 45 pg/ml, preferably about 50 pg/ml, most preferably about 33.25pg/ml. The cut-off range for IL-113 as a marker for periprosthetic jointinfection with at least 90% sensitivity and specificity is about 15pg/ml-50 pg/ml, preferably about 20 pg/ml-45 pg/ml, preferably about 25pg/ml-40 pg/ml, most preferably about 30 pg/ml-35 pg/ml.

In one embodiment, the system is able to detect IL-8 as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity. The cut-off value (derived by ROC analysis) for IL-8 as amarker is about 6500 pg/ml, preferably about 6600 pg/ml, preferablyabout 6700 pg/ml, preferably about 6800 pg/ml, preferably about 6900pg/ml, preferably about 7000 pg/ml, preferably about 7100 pg/ml, mostpreferably about 6797 pg/ml. The cut-off range for IL-8 as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity is about 6500 pg/ml-7100 pg/ml, preferably about 6600pg/ml-7000 pg/ml, preferably about 6700 pg/ml-6900 pg/ml, mostpreferably about 6725 pg/ml-6860 pg/ml.

In one embodiment, the system is able to detect IL-1β as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity. The cut-off value (derived by ROC analysis) for IL-1β as amarker is about 20 pg/ml, preferably about 9 pg/ml, preferably about 12pg/ml, preferably about 15 pg/ml, preferably about 19 pg/ml, preferablyabout 22 pg/ml, preferably about 25 pg/ml, most preferably about 16.25pg/ml. The cut-off range for IL-1β as a marker for periprosthetic jointinfection with at least 90% sensitivity and specificity is about 4pg/ml-26 pg/ml, preferably about 6 pg/ml-24 pg/ml, preferably about 8pg/ml-22 pg/ml, most preferably about 10 pg/ml-20 pg/ml.

In one embodiment, the system is able to detect CRP as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity. The cut-off value (derived by ROC analysis) for CRP as amarker is about 8000 ng/ml, preferably about 9000 ng/ml, preferablyabout 10000 ng/ml, preferably about 11000 ng/ml, preferably about 12000ng/ml, preferably about 13000 ng/ml, preferably about 14000 ng/ml, mostpreferably about 11412 ng/ml. The cut-off range for CRP as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity is about 8000 ng/ml-15000 ng/ml, preferably about 9000ng/ml-14000 ng/ml, preferably about 10000 ng/ml-13000 ng/ml, mostpreferably about 11000 ng/ml-12000 ng/ml. In one embodiment, the systemis able to detect TNFα as a marker for periprosthetic joint infectionwith at least 90% sensitivity and specificity. The cut-off value(derived by ROC analysis) for TNFα as a marker is about 40 pg/ml,preferably about 50 pg/ml, preferably about 60 pg/ml, preferably about70 pg/ml, preferably about 80 pg/ml, preferably about 90 pg/ml,preferably about 100 pg/ml, most preferably about 66.42 pg/ml. Thecut-off range for TNFα as a marker for periprosthetic joint infectionwith at least 90% sensitivity and specificity is about 46 pg/ml-120pg/ml, preferably about 59 pg/ml-110 pg/ml, preferably about 62pg/ml-100 pg/ml, most preferably about 65 pg/ml-88 pg/ml.

In one embodiment, the system is able to detect IL-6 as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity. The cut-off value (derived by ROC analysis) for IL-6 as amarker is about 2900 pg/ml, preferably about 3000 pg/ml, preferablyabout 3100 pg/ml, preferably about 3200 pg/ml, preferably about 3300pg/ml, preferably about 3400 pg/ml, preferably about 3500 pg/ml, mostpreferably about 3102 pg/ml. The cut-off range for IL-6 (Biorad) as amarker for periprosthetic joint infection with at least 90% sensitivityand specificity is about 2300 pg/ml-3700 pg/ml, preferably about 2400pg/ml-3600 pg/ml, preferably about 2500 pg/ml-3500 pg/ml, mostpreferably about 2615 pg/ml-3400 pg/ml.

In one embodiment, the system is able to detect IL-6 as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity. The cut-off value (derived by ROC analysis) for IL-6 as amarker is about 1000 pg/ml, preferably about 1500 pg/ml, preferablyabout 2000 pg/ml, preferably about 3000 pg/ml, preferably about 4000pg/ml, preferably about 5000 pg/ml, preferably about 6000 pg/ml, mostpreferably about 3472 pg/ml. The cut-off range for IL-6 as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity is about 1600 pg/ml-5900 pg/ml, preferably about 1700pg/ml-5600 pg/ml, preferably about 1800 pg/ml-5300 pg/ml, mostpreferably about 1965 pg/ml-5000 pg/ml.

In one embodiment, the system is able to detect Human neutrophilelastase (HNE) as a marker for periprosthetic joint infection with atleast 90% sensitivity and specificity. The cut-off value (derived by ROCanalysis) for HNE as a marker is about 400 ng/ml, preferably about 450ng/ml, preferably about 500 ng/ml, preferably about 550 ng/ml,preferably about 600 ng/ml, preferably about 650 ng/ml, preferably about700 ng/ml, most preferably about 552.8 ng/ml. The cut-off range for HNEas a marker for periprosthetic joint infection with at least 90%sensitivity and specificity is about 400 ng/ml-700 ng/ml, preferablyabout 450 ng/ml-650 ng/ml, preferably about 500 ng/ml-600 ng/ml, mostpreferably about 521 ng/ml-584 ng/ml.

In one embodiment, the system is able to detect alpha(2)-macroglobulin(a2M) as a marker for periprosthetic joint infection with at least 90%sensitivity and specificity. The cut-off value (derived by ROC analysis)for a2M as a marker is about 40 pg/ml, preferably about 50 pg/ml,preferably about 60 pg/ml, preferably about 70 pg/ml, preferably about80 pg/ml, preferably about 90 pg/ml, preferably about 100 pg/ml, mostpreferably about 73.45 pg/ml. The cut-off range for a2M as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity is about 65 pg/ml-90 pg/ml, preferably about 70 pg/ml-85pg/ml, preferably about 75 pg/ml-80 pg/ml, most preferably about 70pg/ml-76 pg/ml.

In one embodiment, the system is able to detect VEGF as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity. The cut-off value (derived by ROC analysis) for VEGF as amarker is about 1800 pg/ml, preferably about 2100 pg/ml, preferablyabout 2400 pg/ml, preferably about 2700 pg/ml, preferably about 3000pg/ml, preferably about 3300 pg/ml, preferably about 3700 pg/ml, mostpreferably about 2565 pg/ml. The cut-off range for VEGF as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity is about 1900 pg/ml-3700 pg/ml, preferably about 2100pg/ml-3700 pg/ml, preferably about 2300 pg/ml-3500 pg/ml, mostpreferably about 2500 pg/ml-3300 pg/ml.

In one embodiment, the system is able to detect FGF2 as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity. The cut-off value (derived by ROC analysis) for FGF2 as amarker is about 0.5 pg/ml, preferably about 1 pg/ml, preferably about 4pg/ml, preferably about 8 pg/ml, preferably about 12 pg/ml, preferablyabout 16 pg/ml, preferably about 20 pg/ml, most preferably about 2.25pg/ml. The cut-off range for FGF2 as a marker for periprosthetic jointinfection with at least 90% sensitivity and specificity is about 0.4pg/ml-16 pg/ml, preferably about 0.6 pg/ml-16 pg/ml, preferably about0.8 pg/ml-14 pg/ml, most preferably about 1 pg/ml-12 pg/ml.

In one embodiment, the system is able to detect G-CSF as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity. The cut-off value (derived by ROC analysis) for G-CSF as amarker is about 40 pg/ml, preferably about 60 pg/ml, preferably about 80pg/ml, preferably about 100 pg/ml, preferably about 120 pg/ml,preferably about 140 pg/ml, preferably about 160 pg/ml, most preferablyabout 94.35 pg/ml. The cut-off range for G-CSF as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity is about 55 pg/ml-160 pg/ml, preferably about 60 pg/ml-140pg/ml, preferably about 65 pg/ml-120 pg/ml, most preferably about 74pg/ml-100 pg/ml.

In one embodiment, the system is able to detect SKALP as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity. The cut-off value (derived by ROC analysis) for SKALP as amarker is about 1500 pg/ml, preferably about 2000 pg/ml, preferablyabout 2500 pg/ml, preferably about 3000 pg/ml, preferably about 3500pg/ml, preferably about 4000 pg/ml, preferably about 4500 pg/ml, mostpreferably about 3721 pg/ml. The cut-off range for SKALP as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity is about 1500 pg/ml-5000 pg/ml, preferably about 1700pg/ml-4500 pg/ml, preferably about 1900 pg/ml-4000 pg/ml, mostpreferably about 2100 pg/ml-3800 pg/ml.

In one embodiment, the system is able to detect IP-10 as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity. The cut-off value (derived by ROC analysis) for IP-10 as amarker is about 3500 pg/ml, preferably about 4000 pg/ml, preferablyabout 4500 pg/ml, preferably about 5000 pg/ml, preferably about 5500pg/ml, preferably about 6000 pg/ml, preferably about 6500 pg/ml, mostpreferably about 5003 pg/ml. The cut-off range for IP-10 as a marker forperiprosthetic joint infection with at least 90% sensitivity andspecificity is about 3000 pg/ml-7000 pg/ml, preferably about 3500pg/ml-6500 pg/ml, preferably about 4000 pg/ml-6000 pg/ml, mostpreferably about 4500 pg/ml-5800 pg/ml.

In one aspect, the present disclosure relates to immunoassays forassessing (e.g., detecting or quantifying) at least one biomarker ofinterest in a test sample, where the sensitivity of the immunoassay isimproved relative to, specifically is from about three to about fifteentimes, especially, from about two to about ten times, higher than, thesensitivity of conventional immunoassays known in the art.

Controls with respect to the presence or absence of the biomarker ordifferential expression of the biomarker may either be normal (i.e., notinfected or not-inflamed tissue, for example) or samples from knowntypes or stages of infection or inflammation or other disease. Asdescribed elsewhere herein, comparison of the expression patterns of thesample to be tested with those of the controls can be used to diagnosethe disease or disorder. In this context, the control or control groupis used for purposes of establishing initial cutoffs for the systems andassay of the invention. Therefore, mere detection of a biomarker of theinvention without the requirement of comparison to a control group candiagnose the disease or disorder in the joint. In this manner, thesystem according to the present invention may be used for qualitative(yes/no answer); semi-quantitative (−/+/++/+++/++++) or quantitativeanswer.

Changes in the levels of expression of the biomarker are associated withpathogenesis. Thus, changes in the expression levels of particularbiomarkers serve as signposts for the presence of and progression ofinfection in the joint. For example, it is the differences in expressionof particular biomarkers that are used to determine whether theinfection in the joint is caused by an aseptic or spetic inflammation.

Disease

The incidence of prosthetic joint infection is higher after a revisionarthroplasty which may be due to the long operation time, scarformation, or recrudescence of unrecognized infection present at theinitial surgery. In certain cases where antibiotic treatment is noteffective, it may mean removing the implant outright, and cleaning theimplant and infected area before replacing the joint, which is costly,both in terms of expenses, time and the patient's condition. Theprocedure involves a surgical incision, drainage of the area, hardwareremoval and debridement of all devitalized tissue in conjunction withlong term bed rest, pharmacological treatment followed by replacement ofthe joint.

In one embodiment of the invention, detection of a marker in a sampleidentifies a subject from which the sample was obtained, as having ornot having a particular pathology. For example, the invention providesthe ability to detect a marker in a synovial fluid sample, whereindetection of the marker identifies whether inflammation in the joint ofthe subject is caused by an infection not.

In one embodiment, the invention provides a system for quicklydiagnosing whether the inflammation in the joint is caused by aninfection or not. Determination of the source of inflammation enablesthe physician to apply the appropriate therapy to ameliorate theinflammation. For example, if the subject has a bacterial infection inthe joint, the patient is treated with anti-bacterial. Alternatively, ifthe diagnosis indicates that the inflammation is not caused by abacterial infection, the physician can apply the appropriate type oftherapy to treat aseptic inflammation.

Detecting a Biomarker

The concentration of the biomarker in a sample may be determined by anysuitable assay. A suitable assay may include one or more of thefollowing methods, an enzyme assay, an immunoassay, mass spectrometry,chromatography, electrophoresis or an antibody microarray, or anycombination thereof. Thus, as would be understood by one skilled in theart, the system and methods of the invention may include any methodknown in the art to detect a biomarker in a sample.

The invention described herein also relates to methods for a multiplexanalysis platform. In one embodiment, the method comprises an analyticalmethod for multiplexing analytical measurements of markers. In anotherembodiment, the method comprises a set of compatible analyticalstrategies for multiplex measurements of markers and/or metabolites insynovial fluid.

In one embodiment, the sample of the invention is a biological sample.The biological sample can originate from solid or fluid samples. Thesample of the invention may comprise synovial fluid, urine, whole blood,blood serum, blood plasma, sweat, cerebrospinal fluid, mucous, saliva,lymph, bronchial aspirates, milk and the like. Preferably the sample issynovial fluid.

Immunoassays

In one embodiment, the systems and methods of the invention can beperformed in the form of various immunoassay formats, which are wellknown in the art. Immunoassays, in their most simple and direct sense,are binding assays involving binding between antibodies and antigen.Many types and formats of immunoassays are known and all are suitablefor detecting the disclosed biomarkers. Examples of immunoassays areenzyme linked immunosorbent assays (ELISAs), enzyme linked immunospotassay (ELISPOT), radioimmunoassays (RIA), radioimmune precipitationassays (RIPA), immunobead capture assays, Western blotting, dotblotting, gel-shift assays, Flow cytometry, protein arrays, multiplexedbead arrays, magnetic capture, in vivo imaging, fluorescence resonanceenergy transfer (FRET), fluorescence recovery/localization afterphotobleaching (FRAP/FLAP), a sandwich assay, a competitive assay, animmunoassay using a biosensor, an immunoprecipitation assay, anagglutination assay, a turbidity assay, a nephlelometric assay, etc.

In general, immunoassays involve contacting a sample suspected ofcontaining a molecule of interest (such as the disclosed biomarkers)with an antibody to the molecule of interest or contacting an antibodyto a molecule of interest (such as antibodies to the disclosedbiomarkers) with a molecule that can be bound by the antibody, as thecase may be, under conditions effective to allow the formation ofimmunocomplexes. Contacting a sample with the antibody to the moleculeof interest or with the molecule that can be bound by an antibody to themolecule of interest under conditions effective and for a period of timesufficient to allow the formation of immune complexes (primary immunecomplexes) is generally a matter of simply bringing into contact themolecule or antibody and the sample and incubating the mixture for aperiod of time long enough for the antibodies to form immune complexeswith, i.e., to bind to, any molecules (e.g., antigens) present to whichthe antibodies can bind. In many forms of immunoassay, thesample-antibody composition, such as a tissue section, ELISA plate, dotblot or Western blot, can then be washed to remove any non-specificallybound antibody species, allowing only those antibodies specificallybound within the primary immune complexes to be detected.

Immunoassays can include methods for detecting or quantifying the amountof a molecule of interest (such as the disclosed biomarkers or theirantibodies) in a sample, which methods generally involve the detectionor quantitation of any immune complexes formed during the bindingprocess. In general, the detection of immunocomplex formation is wellknown in the art and can be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any radioactive, fluorescent, biological orenzymatic tags or any other known label. See, for example, U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149and 4,366,241, each of which is incorporated herein by reference in itsentirety and specifically for teachings regarding immunodetectionmethods and labels.

As used herein, a label can include a fluorescent dye, a member of abinding pair, such as biotin/streptavidin, a metal (e.g., gold), or anepitope tag that can specifically interact with a molecule that can bedetected, such as by producing a colored substrate or fluorescence.Substances suitable for detectably labeling proteins include fluorescentdyes (also known herein as fluorochromes and fluorophores) and enzymesthat react with colorometric substrates (e.g., horseradish peroxidase).The use of fluorescent dyes is generally preferred in the practice ofthe invention as they can be detected at very low amounts. Furthermore,in the case where multiple antigens are reacted with a single array,each antigen can be labeled with a distinct fluorescent compound forsimultaneous detection. Labeled spots on the array are detected using afluorimeter, the presence of a signal indicating an antigen bound to aspecific antibody.

Fluorophores are compounds or molecules that luminesce. Typicallyfluorophores absorb electromagnetic energy at one wavelength and emitelectromagnetic energy at a second wavelength.

There are two main types of immunoassays, homogeneous and heterogeneous.In homogeneous immunoassays, both the immunological reaction between anantigen and an antibody and the detection are carried out in ahomogeneous reaction. Heterogeneous immunoassays include at least oneseparation step, which allows the differentiation of reaction productsfrom unreacted reagents. A variety of immunoassays can be used to detectone or more of the proteins disclosed or incorporated by referenceherein.

ELISA is a heterogeneous immunoassay, which can be used in the methodsdisclosed herein. The assay can be used to detect protein antigens invarious formats. In the “sandwich” format the antigen being assayed isheld between two different antibodies. In this method, a solid surfaceis first coated with a solid phase antibody. The test sample, containingthe antigen (e.g., a diagnostic protein), or a composition containingthe antigen, such as a synovial fluid sample from a subject of interest,is then added and the antigen is allowed to react with the boundantibody. Any unbound antigen is washed away. A known amount ofenzyme-labeled antibody is then allowed to react with the bound antigen.Any excess unbound enzyme-linked antibody is washed away after thereaction. The substrate for the enzyme used in the assay is then addedand the reaction between the substrate and the enzyme produces a colorchange. The amount of visual color change is a direct measurement ofspecific enzyme-conjugated bound antibody, and consequently the antigenpresent in the sample tested.

ELISA can also be used as a competitive assay. In the competitive assayformat, the test specimen containing the antigen to be determined ismixed with a precise amount of enzyme-labeled antigen and both competefor binding to an anti-antigen antibody attached to a solid surface.Excess free enzyme-labeled antigen is washed off before the substratefor the enzyme is added. The amount of color intensity resulting fromthe enzyme-substrate interaction is a measure of the amount of antigenin the sample tested. A heterogeneous immunoassay, such as an ELISA, canbe used to detect any of the proteins disclosed or incorporated byreference herein.

Homogeneous immunoassays include, for example, the Enzyme MultipliedImmunoassay Technique (EMIT), which typically includes a biologicalsample comprising the biomarkers to be measured, enzyme-labeledmolecules of the biomarkers to be measured, specific antibody orantibodies binding the biomarkers to be measured, and a specific enzymechromogenic substrate. In a typical EMIT, excess of specific antibodiesis added to a biological sample. If the biological sample contains theproteins to be detected, such proteins bind to the antibodies. Ameasured amount of the corresponding enzyme-labeled proteins is thenadded to the mixture. Antibody binding sites not occupied by moleculesof the protein in the sample are occupied with molecules of the addedenzyme-labeled protein. As a result, enzyme activity is reduced becauseonly free enzyme-labeled protein can act on the substrate. The amount ofsubstrate converted from a colorless to a colored form determines theamount of free enzyme left in the mixture. A high concentration of theprotein to be detected in the sample causes higher absorbance readings.Less protein in the sample results in less enzyme activity andconsequently lower absorbance readings. Inactivation of the enzyme labelwhen the antigen-enzyme complex is antibody-bound makes the EMIT auseful system, enabling the test to be performed without a separation ofbound from unbound compounds as is necessary with other immunoassaymethods. A homogenous immunoassay, such as an EMIT, can be used todetect any of the proteins disclosed or incorporated by referenceherein.

In many immunoassays, as described elsewhere herein, detection ofantigen is made with the use of antigens specific antibodies as detectormolecules. However, immunoassays and the systems and methods of thepresent invention are not limited to the use of antibodies as detectormolecules. Any substance that can bind or capture the antigen within agiven sample may be used. Aside from antibodies, suitable substancesthat can also be used as detector molecules include but are not limitedto enzymes, peptides, proteins, and nucleic acids. Further, there aremany detection methods known in the art in which the captured antigenmay be detected. In some assays, enzyme-linked antibodies produce acolor change. In other assays, detection of the captured antigen is madethrough detecting fluorescent, luminescent, chemiluminescent, orradioactive signals. The system and methods of the current invention isnot limited to the particular types of detectable signals produced in animmunoassay.

Immunoassay kits are also included in the invention. These kits include,in separate containers (a) monoclonal antibodies having bindingspecificity for the polypeptides used in the diagnosis of inflammationor the source of inflammation; and (b) and anti-antibodyimmunoglobulins. This immunoassay kit may be utilized for the practiceof the various methods provided herein. The monoclonal antibodies andthe anti-antibody immunoglobulins can be provided in an amount of about0.001 mg to 100 grams, and more preferably about 0.01 mg to 1 gram. Theanti-antibody immunoglobulin may be a polyclonal immunoglobulin, proteinA or protein G or functional fragments thereof, which may be labeledprior to use by methods known in the art. In several embodiments, theimmunoassay kit includes two, three or four of: antibodies thatspecifically bind a protein disclosed or incorporated herein.

In one embodiment, the immunoassay kit of the invention can comprise (a)a sample pad, (b) a conjugated label pad, the conjugated label padhaving a detectable label, a portion of the conjugated label pad and aportion of the sample pad forming a first interface, (c) a lateral-flowassay comprising a membrane, a portion of the membrane and a portion ofthe conjugated label pad forming a second interface, and (d) at leastone antibody bound to the membrane, the first interface allowing fluidto flow from the sample pad to the conjugated label pad and contact thedetectable label wherein the biomarker present in the sample forms anbiomarker-conjugated label complex, the second interface allowing fluidto flow from the conjugated label pad to the membrane and to contact theat least one membrane-bound antibody to form to an biomarker-antibodycomplex and cause the detectable label to form a detectable signal.

Biosensors

In one embodiment, the biomarkers of the invention are detected usingbiosensors, e.g. with sensor systems with amperometric, electrochemical,potentiometric, conductimetric, impedance, magnetic, optical, acousticor thermal transducers.

Generally, biosensors include a biosensor recognition element which caninclude proteins, nucleic acids, antibodies, etc. that bind to aparticular biomarker and a transducer which converts a molecular signal(i.e. binding of biomarker to recognition element) into an electric ordigital signal that can be quantified, displayed, and analyzed.Biosensors may also include a reader device which translates the signalinto a user-friendly display of the results. Examples of potentialcomponents that comprise an exemplary biosensor are described inBohunicky et al. (2011, Nanotechnology Science and Applications, 4:1-10), which is hereby incorporated by reference in its entirety.

A biosensor may incorporate a physical, chemical or biological detectionsystem. In one embodiment, a biosensor is a sensor with a biologicalrecognition system, e.g. based on a nucleic acid, such as anoligonucleotide probe or aptamer, or a protein such as an enzyme,binding protein, receptor protein, transporter protein or antibody. Inone embodiment, the biological recognition system may comprisetraditional immunoassays described elsewhere herein. In another element,the recognition element (e.g. protein, nucleic acid, antibody, etc.) maybe unlabeled and binding of the biomarker to the element is directlyobserved and converted into a signal by the transducer.

The method for detection of the biomarker in a biosensor may compriseimmunological, electrical, thermal, magnetic, optical (e.g. hologram) oracoustic technologies. Using such biosensors, it is possible to detectthe target biomarker at the anticipated concentrations found inbiological samples.

The biosensor may incorporate detection methods and systems as describedherein for detection of the biomarker. Biosensors may employ electrical(e.g. amperometric, potentiometric, conductimetric, or impedancedetection systems), calorimetric (e.g. thermal), magnetic, optical (e.g.hologram, luminescence, fluorescence, colorimetry), or mass change (e.g.piezoelectric, acoustic wave) technologies. In a biosensor according tothe invention the level of one, two, three, or more biomarkers can bedetected by one or more methods selected from: direct, indirect orcoupled enzymatic, spectrophotometric, fluorimetric, luminometric,spectrometric, polarimetric and chromatographic techniques. Particularlypreferred biosensors comprise one or more enzymes used directly orindirectly via a mediator, or using a binding, receptor or transporterprotein, coupled to an electrical, optical, acoustic, magnetic orthermal transducer. Using such biosensors, it is possible to detect thelevel of target biomarkers at the anticipated concentrations found inbiological samples.

In one embodiment of a biosensor, a biomarker of the invention can bedetected using a biosensor incorporating technologies based on “smart”holograms, or high frequency acoustic systems, such systems areparticularly amenable to “bar code” or array configurations. In smarthologram sensors (Smart Holograms Ltd, Cambridge, UK), a holographicimage is stored in a thin polymer film that is sensitized to reactspecifically with the biomarker. On exposure, the biomarker reacts withthe polymer leading to an alteration in the image displayed by thehologram. The test result read-out can be a change in the opticalbrightness, image, color and/or position of the image. For qualitativeand semi-quantitative applications, a sensor hologram can be read byeye, thus removing the need for detection equipment. A simple colorsensor can be used to read the signal when quantitative measurements arerequired. Opacity or color of the sample does not interfere withoperation of the sensor. The format of the sensor allows multiplexingfor simultaneous detection of several substances. Reversible andirreversible sensors can be designed to meet different requirements, andcontinuous monitoring of a particular biomarker of interest is feasible.

Biosensors to detect the biomarker of the invention may includeacoustic, surface plasmon resonance, holographic and microengineeredsensors. Imprinted recognition elements, thin film transistortechnology, magnetic acoustic resonator devices and other novelacousto-electrical systems may be employed in biosensors for detectionof the biomarkers of the invention.

Suitably, biosensors for detection of the biomarker of the invention arecoupled, i.e. they combine biomolecular recognition with appropriatemeans to convert detection of the presence, or quantitation, of thebiomarker in the sample into a signal. Biosensors can be adapted for“alternate site” diagnostic testing, e.g. in the ward, outpatients'department, surgery, home, field and workplace.

Methods involving detection and/or quantification of the biomarker ofthe invention can be performed using bench-top instruments, or can beincorporated onto disposable, diagnostic or monitoring platforms thatcan be used in a non-laboratory environment, e.g. in the physician'soffice or at the patient's bedside.

Mass Spectrometry and Chromatography

In one embodiment, the systems and methods of the invention can beperformed in the form of various mass spectrometry (MS) orchromatography formats, which are well known in the art. As such, thelevels of biomarkers present in a sample can be determined by massspectrometry. Generally, any mass spectrometric techniques that canobtain precise information on the mass of peptides, and preferably alsoon fragmentation and/or (partial) amino acid sequence of selectedpeptides, are useful herein. Suitable peptide MS techniques and systemsare well-known per se (see, e.g., Methods in Molecular Biology, vol.146: “Mass Spectrometry of Proteins and Peptides”, by Chapman, ed.,Humana Press 2000, ISBN 089603609x; Biemann 1990. Methods Enzymol 193:455-79; or Methods in Enzymology, vol. 402: “Biological MassSpectrometry”, by Burlingame, ed., Academic Press 2005, ISBN9780121828073) and may be used herein.

The terms “mass spectrometry” or “MS” as used herein refer to methods offiltering, detecting, and measuring ions based on their mass-to-chargeratio, or “m/z.” In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). For examples see U.S. Pat. Nos. 6,204,500,6,107,623, 6,268,144, 6,124,137; Wright et al., 1999, Prostate Cancerand Prostatic Diseases 2: 264-76; Merchant et al., 2000, Electrophoresis21: 1164-67, each of which is hereby incorporated by reference in itsentirety, including all tables, figures, and claims. Mass spectrometrymethods are well known in the art and have been used to quantify and/oridentify biomolecules, such as proteins and hormones (Li et al., 2000,Tibtech. 18:151-160; Starcevic et. al., 2003, J. Chromatography B, 792:197-204; Kushnir et. al., 2006, Clin. Chem. 52:120-128; Rowley et al.,2000, Methods 20: 383-397; Kuster et al., 1998, Curr. Opin. StructuralBiol. 8: 393-400). Further, mass spectrometric techniques have beendeveloped that permit at least partial de novo sequencing of isolatedproteins (Chait et al., 1993, Science, 262:89-92; Keough et al., 1999,Proc. Natl. Acad. Sci. USA. 96:7131-6; Bergman, 2000, EXS 88:133-44).Various methods of ionization are known in the art. For examples,Atmospheric Pressure Chemical Ionization (APCI) Chemical Ionization (CI)Electron Impact (EI) Electrospray Ionization (ESI) Fast Atom Bombardment(FAB) Field Desorption/Field Ionization (FD/FI) Matrix Assisted LaserDesorption Ionization (MALDI) and Thermospray Ionization (TSP).

The levels of biomarkers present in a sample can be determined by MSsuch as matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF) MS; MALDI-TOF post-source-decay (PSD); MALDI-TOF/TOF;surface-enhanced laser desorption/ionization time-of-flight massspectrometry (SELDI-TOF) MS; tandem mass spectrometry (e.g., MS/MS,MS/MS/MS etc.); electrospray ionization mass spectrometry (ESI-MS);ESI-MS/MS; ESI-MS/(MS)n (n is an integer greater than zero); ESI 3D orlinear (2D) ion trap MS; ESI triple quadrupole MS; ESI quadrupoleorthogonal TOF (Q-TOF); ESI Fourier transform MS systems;desorption/ionization on silicon (DIOS); secondary ion mass spectrometry(SIMS); atmospheric pressure chemical ionization mass spectrometry(APCI-MS); APCI-MS/MS; APCI-(MS)^(n); atmospheric pressurephotoionization mass spectrometry (APPI-MS); APPI-MS/MS; APPI-(MS)^(n);liquid chromatography-mass spectrometry (LC-MS), gas chromatography-massspectrometry (GC-MS); high performance liquid chromatography-massspectrometry (HPLC-MS); capillary electrophoresis-mass spectrometry; andnuclear magnetic resonance spectrometry. Peptide ion fragmentation intandem MS (MS/MS) arrangements may be achieved using manners establishedin the art, such as, e.g., collision induced dissociation (CID). See forexample, U.S. Patent Application Nos: 20030199001, 20030134304,20030077616, which are herein incorporated by reference in theirentirety. Such techniques may be used for relative and absolutequantification and also to assess the ratio of the biomarker accordingto the invention with other biomarkers that may be present. Thesemethods are also suitable for clinical screening, prognosis, monitoringthe results of therapy, identifying patients most likely to respond to aparticular therapeutic treatment, for drug screening and development,and identification of new targets for drug treatment.

In certain embodiments, a gas phase ion spectrophotometer is used. Inother embodiments, laser-desorption/ionization mass spectrometry is usedto analyze the sample. Modern laser desorption/ionization massspectrometry (“LDI-MS”) can be practiced in two main variations: matrixassisted laser desorption/ionization (“MALDI”) mass spectrometry andsurface-enhanced laser desorption/ionization (“SELDI”). In MALDI, theanalyte is mixed with a solution containing a matrix, and a drop of theliquid is placed on the surface of a substrate. The matrix solution thenco-crystallizes with the biological molecules. The substrate is insertedinto the mass spectrometer. Laser energy is directed to the substratesurface where it desorbs and ionizes the biological molecules withoutsignificantly fragmenting them. See, e.g., U.S. Pat. No. 5,118,937, andU.S. Pat. No. 5,045,694. In SELDI, the substrate surface is modified sothat it is an active participant in the desorption process. In onevariant, the surface is derivatized with adsorbent and/or capturereagents that selectively bind the biomarker of interest. In anothervariant, the surface is derivatized with energy absorbing molecules thatare not desorbed when struck with the laser. In another variant, thesurface is derivatized with molecules that bind the protein of interestand that contain a photolytic bond that is broken upon application ofthe laser. SELDI is a powerful tool for identifying a characteristic“fingerprint” of proteins and peptides in body fluids and tissues for agiven condition, e.g. drug treatments and diseases. This technologyutilizes protein chips to capture proteins/peptides and a time-of-flightmass spectrometer (tof-MS) to quantitate and calculate the mass ofcompounds ranging from small molecules and peptides of less than 1,000Da up to proteins of 500 kDa. Quantifiable differences inprotein/peptide patterns can be statistically evaluated using automatedcomputer programs which represent each protein/peptide measured in thebiofluid spectrum as a coordinate in multi-dimensional space. The SELDIsystem also has a capability of running hundreds of samples in a singleexperiment. In addition, all the signals from SELDI mass spectrometryare derived from native proteins/peptides (unlike some other proteomicstechnologies which require protease digestion), thus directly reflectingthe underlying physiology of a given condition.

In MALDI and SELDI, the derivatizing agent generally is localized to aspecific location on the substrate surface where the sample is applied.See, e.g., U.S. Pat. No. 5,719,060 and WO 98/59361. The two methods canbe combined by, for example, using a SELDI affinity surface to capturean analyte and adding matrix-containing liquid to the captured analyteto provide the energy absorbing material. For additional informationregarding mass spectrometers, see, e.g., Principles of InstrumentalAnalysis, 3rd edition, Skoog, Saunders College Publishing, Philadelphia,1985; and Kirk-Othmer Encyclopedia of Chemical Technology, 4th ed. Vol.15 (John Wiley & Sons, New York 1995), pp. 1071-1094. Detection andquantification of the biomarker will typically depend on the detectionof signal intensity. For example, in certain embodiments, the signalstrength of peak values from spectra of a first sample and a secondsample can be compared (e.g., visually, by computer analysis etc.), todetermine the relative amounts of particular biomarker. Softwareprograms such as the Biomarker Wizard program (Ciphergen Biosystems,Inc., Fremont, Calif.) can be used to aid in analyzing mass spectra. Themass spectrometers and their techniques are well known to those of skillin the art.

In an embodiment, detection and quantification of biomarkers by massspectrometry may involve multiple reaction monitoring (MRM), such asdescribed among others by Kuhn et al. 2004 (Proteomics 4: 1175-86).

In an embodiment, MS peptide analysis methods may be advantageouslycombined with upstream peptide or protein separation or fractionationmethods, such as for example with the chromatographic and other methodsdescribed herein below.

Chromatography can also be used for measuring biomarkers. As usedherein, the term “chromatography” encompasses methods for separatingchemical substances, referred to as such and vastly available in theart. In a preferred approach, chromatography refers to a process inwhich a mixture of chemical substances (analytes) carried by a movingstream of liquid or gas (“mobile phase”) is separated into components asa result of differential distribution of the analytes, as they flowaround or over a stationary liquid or solid phase (“stationary phase”),between said mobile phase and said stationary phase. The stationaryphase may be usually a finely divided solid, a sheet of filter material,or a thin film of a liquid on the surface of a solid, or the like.Chromatography is also widely applicable for the separation of chemicalcompounds of biological origin, such as, e g, amino acids, proteins,fragments of proteins or peptides, etc.

Chromatography as used herein may be preferably columnar (i.e., whereinthe stationary phase is deposited or packed in a column), preferablyliquid chromatography, and yet more preferably high-performance liquidchromatograph? (HPLC). While particulars of chromatography are wellknown in the art, for further guidance see, e.g., Meyer M., 1998, ISBN:047198373X, and “Practical HPLC Methodology and Applications”,Bidlingmeyer, B. A., John Wiley & Sons Inc., 1993.

Exemplary types of chromatography include, without limitation, HPLC,normal phase HPLC (NP-HPLC), reversed phase HPLC (RP-HPLC), ion exchangechromatography (IEC), such as cation or anion exchange chromatography,hydrophilic interaction chromatography (HILIC), hydrophobic interactionchromatography (HIC), size exclusion chromatography (SEC) including gelfiltration chromatography or gel permeation chromatography,chromatofocusing, affinity chromatography such as immuno-affinity,immobilized metal affinity chromatography, and the like.

In an embodiment, chromatography, including single-, two- ormore-dimensional chromatography, may be used as a peptide fractionationmethod in conjunction with a further peptide analysis method, such asfor example, with a downstream mass spectrometry analysis as describedelsewhere in this specification.

Further peptide or polypeptide separation, identification orquantification methods may be used, optionally in conjunction with anyof the above described analysis methods, for measuring biomarkers in thepresent disclosure. Such methods include, without limitation, chemicalextraction partitioning, isoelectric focusing (IEF) including capillaryisoelectric focusing (CIEF), capillary isotachophoresis (CITP),capillary electrochromatography (CEC), and the like, one-dimensionalpolyacrylamide gel electrophoresis (PAGE), two-dimensionalpolyacrylamide gel electrophoresis (2D-PAGE), capillary gelelectrophoresis (CGE), capillary zone electrophoresis (CZE), micellarelectrokinetic chromatography (MEKC), free flow electrophoresis (FFE),etc.

Point-of-Use Devices

Point-of-use analytical tests have been developed for the routineidentification or monitoring of health-related conditions (such aspregnancy, cancer, endocrine disorders, infectious diseases or drugabuse) using a variety of biological samples (such as urine, serum,plasma, blood, saliva). Some of the point-of-use assays are based onhighly specific interactions between specific binding pairs, such asantigen/antibody, hapten/antibody, lectin/carbohydrate,apoprotein/cofactor and biotin/(strept)avidin. In some point-of usedevices, assays are performed with test strips in which a specificbinding pair member is attached to a mobilizable material (such as ametal sol or beads made of latex or glass) or an immobile substrate(such as glass fibers, cellulose strips or nitrocellulose membranes).Other point-of use devices may comprise optical biosensors, photometricbiosensors, electrochemical biosensor, or other types of biosensors.Suitable biosensors in point-of-use devices for performing methods ofthe invention include “cards” or “chips” with optical or acousticreaders. Biosensors can be configured to allow the data collected to beelectronically transmitted to the physician for interpretation and thuscan form the basis for e-medicine, where diagnosis and monitoring can bedone without the need for the patient to be in proximity to a physicianor a clinic.

Detection of a biomarker in a synovial fluid can be carried out using asample capture device, such as a lateral flow device (for example alateral flow test strip) that allows detection of one or morebiomarkers, such as those described herein.

The test strips of the present invention include a flow path from anupstream sample application area to a test site. For example, the flowpath can be from a sample application area through a mobilization zoneto a capture zone. The mobilization zone may contain a mobilizablemarker that interacts with an analyte or analyte analog, and the capturezone contains a reagent that binds the analyte or analyte analog todetect the presence of an analyte in the sample.

Examples of migration assay devices, which usually incorporate withinthem reagents that have been attached to colored labels, therebypermitting visible detection of the assay results without addition offurther substances are found, for example, in U.S. Pat. No. 4,770,853(incorporated herein by reference). There are a number of commerciallyavailable lateral-flow type tests and patents disclosing methods for thedetection of large analytes (MW greater than 1,000 Daltons) as theanalyte flows through multiple zones on a test strip. Examples are foundin U.S. Pat. Nos. 5,229,073, 5,591,645; 4,168,146; 4,366,241; 4,855,240;4,861,711; 5,120,643 (each of which are herein incorporated byreference). Multiple zone lateral flow test strips are disclosed in U.S.Pat. Nos. 5,451,504, 5,451,507, and U.S. Pat. No. 5,798,273(incorporated by reference herein). U.S. Pat. No. 6,656,744(incorporated by reference) discloses a lateral flow test strip in whicha label binds to an antibody through a streptavidin-biotin interaction.

Flow-through type assay devices were designed, in part, to obviate theneed for incubation and washing steps associated with dipstick assays.Flow-through immunoassay devices involve a capture reagent (such as oneor more antibodies) bound to a porous membrane or filter to which aliquid sample is added. As the liquid flows through the membrane, targetanalyte (such as protein) binds to the capture reagent. The addition ofsample is followed by (or made concurrent with) addition of detectorreagent, such as labeled antibody (e.g., gold-conjugated or coloredlatex particle-conjugated protein). Alternatively, the detector reagentmay be placed on the membrane in a manner that permits the detector tomix with the sample and thereby label the analyte. The visual detectionof detector reagent provides an indication of the presence of targetanalyte in the sample. Representative flow-through assay devices aredescribed in U.S. Pat. Nos. 4,246,339; 4,277,560; 4,632,901; 4,812,293;4,920,046; and 5,279,935; U.S. Patent Application Publication Nos.20030049857 and 20040241876; and WO 08/030,546. Migration assay devicesusually incorporate within them reagents that have been attached tocolored labels, thereby permitting visible detection of the assayresults without addition of further substances. See, for example, U.S.Pat. No. 4,770,853; PCT Publication No. WO 88/08534.

There are a number of commercially available lateral flow type tests andpatents disclosing methods for the detection of large analytes (MWgreater than 1,000 Daltons). U.S. Pat. No. 5,229,073 describes asemiquantitative competitive immunoassay lateral flow method formeasuring plasma lipoprotein levels. This method utilizes a plurality ofcapture zones or lines containing immobilized antibodies to bind boththe labeled and free lipoprotein to give a semi-quantitative result. Inaddition, U.S. Pat. No. 5,591,645 provides a chromatographic test stripwith at least two portions. The first portion includes a movable tracerand the second portion includes an immobilized binder capable of bindingto the analyte. Additional examples of lateral flow tests for largeanalytes are disclosed in the following patent documents: U.S. Pat. Nos.4,168,146; 4,366,241; 4,855,240; 4,861,711; and 5,120,643; WO 97/06439;WO 98/36278; and WO 08/030,546.

Devices described herein generally include a strip of absorbent material(such as a microporous membrane), which, in some instances, can be madeof different substances each joined to the other in zones, which may beabutted and/or overlapped. In some examples, the absorbent strip can befixed on a supporting non-interactive material (such as nonwovenpolyester), for example, to provide increased rigidity to the strip.Zones within each strip may differentially contain the specific bindingpartner(s) and/or other reagents required for the detection and/orquantification of the particular analyte being tested for, for example,one or more proteins disclosed herein. Thus these zones can be viewed asfunctional sectors or functional regions within the test device.

In general, a fluid sample is introduced to the strip at the proximalend of the strip, for instance by dipping or spotting. A sample iscollected or obtained using methods well known to those skilled in theart. The sample containing the particular proteins to be detected may beobtained from any biological source. In a particular example, thebiological source is synovial fluid. The sample may be diluted,purified, concentrated, filtered, dissolved, suspended or otherwisemanipulated prior to assay to optimize the immunoassay results. Thefluid migrates distally through all the functional regions of the strip.The final distribution of the fluid in the individual functional regionsdepends on the adsorptive capacity and the dimensions of the materialsused.

In some embodiments, porous solid supports, such as nitrocellulose,described elsewhere herein are preferably in the form of sheets orstrips. The thickness of such sheets or strips may vary within widelimits, for example, from about 0.01 to 0.5 mm, from about 0.02 to 0.45mm, from about 0.05 to 0.3 mm, from about 0.075 to 0.25 mm, from about0.1 to 0.2 mm, or from about 0.11 to 0.15 mm. The pore size of suchsheets or strips may similarly vary within wide limits, for example fromabout 0.025 to 15 microns, or more specifically from about 0.1 to 3microns; however, pore size is not intended to be a limiting factor inselection of the solid support. The flow rate of a solid support, whereapplicable, can also vary within wide limits, for example from about12.5 to 90 sec/cm (i.e., 50 to 300 sec/4 cm), about 22.5 to 62.5 sec/cm(i.e., 90 to 250 sec/4 cm), about 25 to 62.5 sec/cm (i.e., 100 to 250sec/4 cm), about 37.5 to 62.5 sec/cm (i.e., 150 to 250 sec/4 cm), orabout 50 to 62.5 sec/cm (i.e., 200 to 250 sec/4 cm).

Another common feature to be considered in the use of assay devices is ameans to detect the formation of a complex between an analyte (such asone or more proteins described herein) and a capture reagent (such asone or more antibodies). A detector (also referred to as detectorreagent) serves this purpose. A detector may be integrated into an assaydevice (for example includes in a conjugate pad), or may be applied tothe device from an external source.

A detector may be a single reagent or a series of reagents thatcollectively serve the detection purpose. In some instances, a detectorreagent is a labeled binding partner specific for the analyte (such as agold-conjugated antibody for a particular protein of interest).

In other instances, a detector reagent collectively includes anunlabeled first binding partner specific for the analyte and a labeledsecond binding partner specific for the first binding partner and soforth. Thus, the detector can be a labeled antibody specific for aprotein described herein. The detector can also be an unlabeled firstantibody specific for the protein of interest and a labeled secondantibody that specifically binds the unlabeled first antibody. In eachinstance, a detector reagent specifically detects bound analyte of ananalyte-capture reagent complex and, therefore, a detector reagentpreferably does not substantially bind to or react with the capturereagent or other components localized in the analyte capture area. Suchnon-specific binding or reaction of a detector may provide a falsepositive result. Optionally, a detector reagent can specificallyrecognize a positive control molecule (such as a non-specific human IgGfor a labeled Protein A detector, or a labeled Protein G detector, or alabeled anti-human Ab(Fc)) that is present in a secondary capture area.

Flow-Through Device Construction and Design

A flow-through device involves a capture reagent (such as one or moreantibodies) immobilized on a solid support, typically, microtiter plateor a membrane (such as, nitrocellulose, nylon, or PVDF). In a simplerepresentative format, the membrane of a flow-through device is placedin functional or physical contact with an absorbent layer, which acts asa reservoir to draw a fluid sample through the membrane. Optionally,following immobilization of a capture reagent, any remainingprotein-binding sites on the membrane can be blocked (either before orconcurrent with sample administration) to minimize nonspecificinteractions.

In operation of a flow-through device, a fluid sample is placed incontact with the membrane. Typically, a flow-through device alsoincludes a sample application area (or reservoir) to receive andtemporarily retain a fluid sample of a desired volume. The sample passesthrough the membrane matrix. In this process, an analyte in the sample(such as one or more protein, for example, one or more proteinsdescribed herein) can specifically bind to the immobilized capturereagent (such as one or more antibodies). Where detection of ananalyte-capture reagent complex is desired, a detector reagent (such aslabeled antibodies that specifically bind one or more proteins) can beadded with the sample or a solution containing a detector reagent can beadded subsequent to application of the sample. If an analyte isspecifically bound by capture reagent, a characteristic attributable tothe particular detector reagent can be observed on the surface of themembrane. Optional wash steps can be added at any time in the process,for instance, following application of the sample, and/or followingapplication of a detector reagent.

Lateral Flow Device Construction and Design

Lateral flow devices are commonly known in the art. Briefly, a lateralflow device is an analytical device having as its essence a test strip,through which flows a test sample fluid that is suspected of containingan analyte of interest. The test fluid and any suspended analyte canflow along the strip to a detection zone in which the analyte (ifpresent) interacts with a capture agent and a detection agent toindicate a presence, absence and/or quantity of the analyte.

Numerous lateral flow analytical devices have been disclosed, andinclude those shown in U.S. Pat. Nos. 4,313,734; 4,435,504; 4,775,636;4,703,017; 4,740,468; 4,806,311; 4,806,312; 4,861,711; 4,855,240;4,857,453; 4,943,522; 4,945,042; 4,496,654; 5,001,049; 5,075,078;5,126,241; 5,451,504; 5,424,193; 5,712,172; 6,555,390; 6,258,548;6,699,722; 6,368,876 and 7,517,699, each of which is incorporated byreference.

Many lateral flow devices are one-step lateral flow assays in which abiological fluid is placed in a sample area on a bibulous strip (thoughnon-bibulous materials can be used, and rendered bibulous, e.g., byapplying a surfactant to the material), and allowed to migrate along thestrip until the liquid comes into contact with a specific bindingpartner (such as an antibody) that interacts with an analyte (such asone or more proteins) in the liquid. Once the analyte interacts with thebinding partner, a signal (such as a fluorescent or otherwise visibledye) indicates that the interaction has occurred. Multiple discretebinding partners (such as antibodies) can be placed on the strip (forexample in parallel lines) to detect multiple analytes (such as two ormore proteins) in the liquid. The test strips can also incorporatecontrol indicators, which provide a signal that the test has adequatelybeen performed, even if a positive signal indicating the presence (orabsence) of an analyte is not seen on the strip.

Lateral flow devices have a wide variety of physical formats that areequally well known in the art. Any physical format that supports and/orhouses the basic components of a lateral flow device in the properfunction relationship is contemplated by this disclosure.

The basic components of a particular embodiment of a lateral flow deviceare illustrated in FIGS. 1 and 2 which comprise a sample pad, aconjugate pad, a migration membrane, and an absorbent pad.

The sample pad (such as the sample pad in FIGS. 1 and 2) is a componentof a lateral flow device that initially receives the sample, and mayserve to remove particulates from the sample. Among the variousmaterials that may be used to construct a sample pad (such as glassfiber, woven fibers, screen, non-woven fibers, cellosic fibers or paper)or a cellulose sample pad may be beneficial if a large bed volume is afactor in a particular application. Sample pads may be treated with oneor more release agents, such as buffers, salts, proteins, detergents,and surfactants. Such release agents may be useful, for example, topromote resolubilization of conjugate-pad constituents, and to blocknon-specific binding sites in other components of a lateral flow device,such as a nitrocellulose membrane. Representative release agentsinclude, for example, trehalose or glucose (1%-5%), PVP or PVA(0.5%-2%), Tween 20 or Triton X-100 (0.1%-1%), casein (1%-2%), SDS(0.02%-5%), and PEG (0.02%-5%).

With respect to the migration membrane, the types of membranes useful ina lateral flow device include but are not limited to nitrocellulose(including pure nitrocellulose and modified nitrocellulose) andnitrocellulose direct cast on polyester support, polyvinylidenefluoride, or nylon).

The conjugate pad (such as conjugate pad in FIGS. 1 and 2) serves to,among other things, hold a detector reagent. Suitable materials for theconjugate pad include glass fiber, polyester, paper, or surface modifiedpolypropylene.

Detector reagent(s) contained in a conjugate pad is typically releasedinto solution upon application of the test sample. A conjugate pad maybe treated with various substances to influence release of the detectorreagent into solution. For example, the conjugate pad may be treatedwith PVA or PVP (0.5% to 2%) and/or Triton X-100 (0.5%). Other releaseagents include, without limitation, hydroxypropylmethyl cellulose, SDS,Brij and β-lactose. A mixture of two or more release agents may be usedin any given application.

With respect to the absorbent pad, the pad acts to increase the totalvolume of sample that enters the device. This increased volume can beuseful, for example, to wash away unbound analyte from the membrane. Anyof a variety of materials is useful to prepare an absorbent pad, forexample, cellulosic filters or paper. In some device embodiments, anabsorbent pad can be paper (i.e., cellulosic fibers). One of skill inthe art may select a paper absorbent pad on the basis of, for example,its thickness, compressibility, manufacturability, and uniformity of bedvolume. The volume uptake of an absorbent made may be adjusted bychanging the dimensions (usually the length) of an absorbent pad.

In operation of the particular embodiment of a lateral flow device, afluid sample containing an analyte of interest, such as one or moreproteins described herein, is applied to the sample pad. In someexamples, the sample may be applied to the sample pad by dipping the endof the device containing the sample pad into the sample (such assynovial) or by applying the sample directly onto the sample pad.

From the sample pad, the sample passes, for instance by capillaryaction, to the conjugate pad. In the conjugate pad, the analyte ofinterest, such as a protein of interest, may bind (or be bound by) amobilized or mobilizable detector reagent, such as an antibody (such asantibody that recognizes one or more of the proteins described herein).For example, a protein analyte may bind to a labeled (e.g.,gold-conjugated or colored latex particle-conjugated) antibody containedin the conjugate pad. The analyte complexed with the detector reagentmay subsequently flow to the test line where the complex may furtherinteract with an analyte-specific binding partner (such as an antibodythat binds a particular protein, an anti-hapten antibody, orstreptavidin), which is immobilized at the proximal test line. In someexamples, a protein complexed with a detector reagent (such asgold-conjugated antibody) may further bind to unlabeled, oxidizedantibodies immobilized at the proximal test line. The formation of acomplex, which results from the accumulation of the label (e.g., gold orcolored latex) in the localized region of the proximal test line, isdetected. The control line may contain an immobilized,detector-reagent-specific binding partner, which can bind the detectorreagent in the presence or absence of the analyte. Such binding at thecontrol line indicates proper performance of the test, even in theabsence of the analyte of interest.

In one embodiment, the control line detects the presence of hyaluronicacid which is a marker for synovial fluid in order to ensure that thetest sample is indeed synovial fluid. Therefore, in this situation, thecontrol line is formed by using antibodies to hyaluronic acid (FIG. 2).If the sample is obtained from anything other than the joint (i.e.blood), no line is formed and the test is deemed to be invalid.

The test results may be visualized directly, or may be measured using areader (such as a scanner). The reader device may detect color,fluorescence, luminescence, radioactivity, or any other detectablemarker derived from the labeled reagent from the readout area (forexample, the test line and/or control line).

In another embodiment of a lateral flow device, there may be a second(or third, fourth, or more) test line located parallel or perpendicular(or in any other spatial relationship) to test line in test result zone(for example test lines to biomarker 1 and biomarker 2 in FIG. 2). Theoperation of this particular embodiment is similar to that describedelsewhere herein with the additional considerations that (i) a seconddetector reagent specific for a second analyte, such as anotherantibody, may also be contained in the conjugate pad, and (ii) thesecond test line will contain a second specific binding partner havingaffinity for a second analyte, such as a second protein in the sample.Similarly, if a third (or more) test line is included, the test linewill contain a third (or more) specific binding partner having affinityfor a third (or more) analyte.

In one embodiment, a comparison of the control line to the test lineyields the test result from the diagnostic system of the invention. Insome instances, a valid result occurs when the control line is detectedat a higher intensity level than the test line. For example, a validresult occurs when the control line is at least 5% or more, for example,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more darker thanthe test line. In some instances, a valid result occurs when the controlline is at least 0.5 fold or more, for example, 1 fold, 2 fold, 3 fold,4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold or more darkerthan the test line.

In one embodiment, the control line is a reference line that insuresthat the test has been run correctly and that the tested sample is notobtained from anything other than the joint (i.e. blood). For example,the system of the invention is useful in the diagnosis of infection in ajoint when the control line is detected at an at least equal intensitythan the test line. Preferably, the control line is detected at higherintensity than the test line. In some instances, if the test line is notat least equal in darkness or intensity as the control line then thetest is said to have an invalid result. If the test line is at leastequal or lighter than the control line then the test is said to have avalid result.

In one embodiment, the control line is a reference line that detects HA,wherein HA is a valid marker for distinguishing synovial fluid fromother bodily fluids (e.g., blood). In some instances, HA is detected byusing aggrecan to detect difference between blood and synovial fluid.This is because aggrecan can effectively bind to HA.

Point of Care Diagnostic and Risk Assessment Systems

The system of the invention can be applied to a point-of-care scenario.U.S. Pat. Nos. 6,267,722, 6,394,952 and 6,867,051 disclose and describesystems for diagnosing and assessing certain medical risks, the contentsof which are incorporated herein. The systems are designed for use onsite at the point of care, where patients are examined and tested, aswell as for operation remote from the site. The systems are designed toaccept input in the form of patient data, including, but not limited tobiochemical test data, physical test data, historical data and othersuch data, and to process and output information, such as data relatingto a medical diagnosis or a disease risk indicator. The patient data maybe contained within the system, such as medical records or history, ormay be input as a signal or image from a medical test or procedure, forexample, immunoassay test data, blood pressure reading, ultrasound,X-ray or MRI, or introduced in any other form. Specific test data can bedigitized, processed and input into the medical diagnosis expert system,where it may be integrated with other patient information. The outputfrom the system is a disease risk index or medical diagnosis.

Point of care testing refers to real time diagnostic testing that can bedone in a rapid time frame so that the resulting test is performedfaster than comparable tests that do not employ this system. Forexample, the exemplified immunoassay disclosed and described herein canbe performed in significantly less time than the corresponding ELISAassay, e.g., in less than half an hour. In addition, point of caretesting refers to testing that can be performed rapidly and on site,such as in a doctor's office, at a bedside, in a stat laboratory,emergency room or other such locales, particularly where rapid andaccurate results are required.

In an exemplary embodiment, a point of care diagnostic and riskassessment system includes a reader for reading patient data, a testdevice designed to be read in the reader, and software for analysis ofthe data. A test strip device in a plastic housing is designed for usewith the reader, optionally including a symbology, such as analphanumeric character bar code or other machine-readable code, andsoftware designed for analysis of the data generated from the test stripare also provided.

In one embodiment, a reader refers to an instrument for detecting and/orquantitating data, such as on test strips. The data may be visible tothe naked eye, but does not need to be visible. Such readers aredisclosed and described in the above-incorporated U.S. Pat. Nos.6,267,722, 6,394,952 and 6,867,051. A reflectance reader refers to aninstrument adapted to read a test strip using reflected light, includingfluorescence, or electromagnetic radiation of any wavelength.Reflectance can be detected using a photodetector or other detector,such as charge coupled diodes (CCD). An exemplary reflectance readerincludes a cassette slot adapted to receive a test-strip, light-emittingdiodes, optical fibers, a sensing head, including means for positioningthe sensing head along the test strip, a control circuit to read thephotodetector output and control the on and off operation of thelight-emitting diodes, a memory circuit for storing raw and/or processeddata, and a photodetector, such as a silicon photodiode detector. Itwill be appreciated that a color change refers to a change in intensityor hue of color or may be the appearance of color where no color existedor the disappearance of color.

In one embodiment, a sample is applied to a diagnostic immunoassay teststrip, and colored or dark bands are produced. The intensity of thecolor reflected by the colored label in the test region (or detectionzone) of the test strip is, for concentration ranges of interest,directly proportional or otherwise correlated with an amount of analytepresent in the sample being tested. The color intensity produced isread, in accordance with the present embodiment, using a reader device,for example, a reflectance reader, adapted to read the test strip. Theintensity of the color reflected by the colored label in the test region(or detection zone) of the test strip is directly proportional to theamount of analyte present in the sample being tested. In other words, adarker colored line in the test region indicates a greater amount ofanalyte, whereas a lighter colored line in the test region indicates asmaller amount of analyte. The color intensity produced, i.e., thedarkness or lightness of the colored line, is read using a readerdevice, for example, a reflectance reader, adapted to read the teststrip.

A reflectance measurement obtained by the reader device is correlated tothe presence and/or quantity of analyte present in the sample. Thereader takes a plurality of readings along the strip, and obtains datathat are used to generate results that are an indication of the presenceand/or quantity of analyte present in the sample. The system maycorrelate such data with the presence of a disorder, condition or riskthereof.

As mentioned elsewhere herein, in addition to reading the test strip,the reader may (optionally) be adapted to read a symbology, such as abar code, which is present on the test strip or housing and encodesinformation relating to the test strip device and/or test result and/orpatient, and/or reagent or other desired information. Typically theassociated information is stored in a remote computer database, but canbe manually stored. Furthermore, the symbology can be imprinted when thedevice is used and the information encoded therein.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1 System for Detecting Biomarkers in Joint Fluid

Experiments were designed to develop a system for collecting andanalyzing joint fluid in a simple and reproducible manner. Analysis ofjoint fluid is ideally suited for immunoassay. Unlike blood, which isthe most commonly tested diagnostic sample, joint fluid does notcirculate. Therefore, joint fluid presents a unique picture of what ishappening in one specific location of the body (i.e. the joint). Beingstationary also prevents dilution of targeted compounds which ensuresthat the concentration of the proteins is in a range easily measurableby immunoassay. Therefore, joint fluid (e.g., synovial fluid) can beused for the analysis of joint pain.

The system of the invention useful for analyzing joint fluid can bebased on a lateral flow system. For example, the lateral flowimmunoassay utilizes strips of cellulose membrane onto which antibodiesand other reagents can be coated. The sample moves along the strip dueto capillary action and reacts with the reagents at different pointsalong the strip. The end result is usually the appearance or absence ofa colored line or spot (FIG. 1). Because a lateral flow assay forbiomarkers can be in the form of a sandwich assay, the signal resultsfrom the formation of an immunocomplex on the membrane.

The advantages of a lateral flow assay include quick results followingapplication of the sample, no instrumentation is required, able toexecute the test and read the result in the field itself, and allows forroom temperature storage.

The system of the invention is useful for detecting biomarkers.Biomarkers are proteins or other cellular components that relatespecifically to injury or to disease and that can be found in bodyfluids such as synovial fluid. A biomarker designates any indicatory(nucleic acids, enzymes, metabolites and other types of molecules:histamines, hormones, proteins, etc.) present in the body as abiological response to disease. The identification and development ofbiomarkers make it possible to perform faster, more accurate diseasediagnostics and help physicians prescribe new treatments for thosepatients who are likely to benefit from them. Tests bases on biomarkersoften contribute to a faster cure and avoid time-consuming and costlyanalyses.

To be specific for injury or disease, the presence of the biomarkers inthe synovial fluid must depend on the diseased state and should not bepresent under normal conditions or in different disease states. The useof synovial fluid provides an optimum medium for the use of biomarkersin diagnosing, for example, the source of joint pain because joint fluidis a “closed” system. In an experiment where the concentrations of IL-6in serum versus synovial fluid from patients with an infected knee werecompared, a 1000× amplification in the synovial fluid was observed.

Unlike with the use of blood where the presence of a biomarker can be aresult from anywhere in the body, the presence of a biomarker insynovial fluid is a result from a local response.

One of the concerns in sample collection is to ensure that the withdrawnsample is indeed synovial fluid. Therefore, in this situation, thecontrol line is formed by using monoclonal antibodies to hyaluronic acid(FIG. 2). If the sample is from anything other than the joint (i.e.serum), no line is formed and the test is deemed invalid.

Example 2 Synovial Fluid Biomarkers for Periprosthetic Infection

Experiments were designed to evaluate biomarker data from a study ofsynovial fluid biomarkers for periprosthetic infection (Deirmangian C etal., 2010, Clin Orthop Relat Res 468:2017-2023) that appeared todiscriminate between infected and aseptic groups. ROC Analysis for AreaUnder the Curve (AUC), which is a measure of the degree of separationbetween the clinical groups at various thresholds, was used to furtherevaluate the biomarker data. Without wishing to be bound by anyparticular theory, it is believed that the higher the AUC value, thebetter the discrimination between the 2 clinical groups, and therebyindicating the predictiveness of the biomarker.

A ROC analysis for selected biomarkers was performed using Prism 5 forMac OS X. The data output was summarized in Tables 1-9 below and a dotplot for each selected biomarker in FIGS. 3-11, respectively.

TABLE 1 Interleukin-1α Parameter Level Cut-off (pg/mL) 1.0 Specificity(%) 97.3 Sensitivity (%) 85.7 NPV (%) 94.7 PPV (%) 92.3 AUC 0.910 95%confidence interval 0.796 to 1.024 P value <0.0001 Mean of asepticsamples (pg/mL) 1.0 Mean of infected samples (pg/mL) 24.9 Fold elevationof infected from aseptic 24.9 samples

TABLE 2 Interleukin-1β Parameter Level Cut-off (pg/mL) 112 Specificity(%) 100.0 Sensitivity (%) 100.0 NPV (%) 100.0 PPV (%) 100.0 AUC 1.00095% confidence interval 1.000 to 1.000 P value <0.0001 Mean of asepticsamples (pg/mL) 8.0 Mean of infected samples (pg/mL) 2067.2 Foldelevation of infected from aseptic 258.4 samples

TABLE 3 Interleukin-6 Parameter Level Cut-off (pg/mL) 13350 Specificity(%) 100.0 Sensitivity (%) 100.0 NPV (%) 100.0 PPV (%) 100.0 AUC 1.00095% confidence interval 1.000 to 1.000 P value <0.0001 Mean of asepticsamples (pg/mL) 2171.7 Mean of infected samples (pg/mL) 59324.8 Foldelevation of infected from aseptic 27.3 samples

TABLE 4 Interleukin-8 Parameter Level Cut-off (pg/mL) 8790 Specificity(%) 97.3 Sensitivity (%) 71.4 NPV (%) 90.0 PPV (%) 90.9 AUC 0.923 95%confidence interval 0.842 to 1.004 P value <0.0001 Mean of asepticsamples (pg/mL) 3402.7 Mean of infected samples (pg/mL) 21238.8 Foldelevation of infected from aseptic 6.2 samples

TABLE 5 Interleukin-10 Parameter Level Cut-off (pg/mL) 10.0 Specificity(%) 89.2 Sensitivity (%) 85.7 NPV (%) 94.3 PPV (%) 75.0 AUC 0.975 95%confidence interval 0.939 to 1.011 P value <0.0001 Mean of asepticsamples (pg/mL) 4.1 Mean of infected samples (pg/mL) 32.6 Fold elevationof infected from aseptic 8.0 samples

TABLE 6 Interleukin-17 Parameter Level Cut-off (pg/mL) 7.2 Specificity(%) 97.3 Sensitivity (%) 85.7 NPV (%) 94.7 PPV (%) 92.3 AUC 0.921 95%confidence interval 0.810 to 1.032 P value <0.0001 Mean of asepticsamples (pg/mL) 2.8 Mean of infected samples (pg/mL) 314.6 Foldelevation of infected from aseptic 112.4 samples

TABLE 7 G-CSF Parameter Level Cut-off (pg/mL) 35.0 Specificity (%) 94.6Sensitivity (%) 100.0 NPV (%) 100.0 PPV (%) 87.5 AUC 0.994 95%confidence interval 0.981 to 1.007 P value <0.0001 Mean of asepticsamples (pg/mL) 10.7 Mean of infected samples (pg/mL) 1283.8 Foldelevation of infected from aseptic 120.0 samples

TABLE 8 VEGF Parameter Level Cut-off (pg/mL) 1500 Specificity (%) 48.6Sensitivity (%) 85.7 NPV (%) 90.0 PPV (%) 61.3 AUC 0.677 95% confidenceinterval 0.528 to 0.825 P value 0.0302 Mean of aseptic samples (pg/mL)2374.5 Mean of infected samples (pg/mL) 3100.6 Fold elevation ofinfected from aseptic 1.3 samples

TABLE 9 SKALP Parameter Level Cut-off (pg/mL) 1880 Specificity (%) 89.2Sensitivity (%) 78.6 NPV (%) 91.7 PPV (%) 73.3 AUC 0.900 95% confidenceinterval 0.814 to 0.9855 P value <0.0001 Mean of aseptic samples (pg/mL)1241.1 Mean of infected samples (pg/mL) 2650.7 Fold elevation ofinfected from aseptic 2.1 samples

The results presented herein demonstrate that the AUC values for theselected biomarkers are high and show desirable discrimination betweenthe 2 clinical groups. The results indicate that the predictiveness ofthese biomarkers is desirable.

Example 3 Pilot Assay for HNP1-3

The results presented herein demonstrate that HNP1-3 is a biomarker forinfection in joint, preferably a replacement joint or periprostheticjoint.

The materials and methods employed in the experiments disclosed hereinare now described.

Briefly, components were sourced from Hycult Biotech Human HNP1-3 ElisaKit, catalog HK317. All reagents were brought to room temperature beforeuse. Dilution buffer was prepared by mixing 10 ml of the 10× dilutionbuffer with 90 ml of deionized H₂O. As per the manufactures suggestion,the buffer mixture was allowed to sit for 10 minutes and inspected forcrystals/particulate before being used. To make the standard/samplebuffer, 90 ml of this dilution buffer was mixed with 10 ml of plasmadiluent.

Synovial fluid and serum samples were diluted in sample buffer at both1:100 and 1:1000. 100 ul of each standard or sample was then added tothe assay plate as discussed elsewhere herein. The plate was covered andincubated at RT for 1 hour. The plate was then washed 3× using thebiotek automated plate washer program “flat nunc plate” with in-housewash buffer of DPBS with 0.05% tween. This automated washer and washbuffer were used for all wash steps.

100 ul/well of the tracer solution was added, the plate covered, and theplate was then incubated at RT for 1 hour. The 3× wash procedure wasrepeated, then 100 ul/well of a streptavidin-peroxidase conjugate wasadded to the plate. The plate was sealed and incubated for 1 hour at RT.After washing 3×, 100 ul/well of TMB substrate was added. The plate wasdeveloped in the dark for 30 minutes, then stopeed using a stopsolution. The assay was then read at 450 nm absorbance using theSpectraMax plate reader with SoftMax software.

The results of the experiments presented in this Example are nowdescribed.

From a clinical standpoint, HNP1-3 showed a clear separation betweenperiprosthetic joint infection positive and negative samples (FIG. 12).These negative samples were sourced from multiple patient types andfluids, including serum, psuedogout, metal on metal (MOM),osteoarthritis (OA), and inflamed but not infected.

With separation between known periprosthetic joint infection positiveand negative samples, including challenging negatives such as pseudogoutand metal on metal, HNP1-3 is a clinically significant biomarker.

The next set of experiments was designed to use an accurate standardcurve in order to interpolate dose concentrations and determine cutoffpoints via ROC analysis. For example, experiments were performed to seekan optimal dilution at which to run synovial fluid samples with theHycult HNP1-3 Elisa kit.

The standard was reconstituted with 0.5 ml of di H₂O, yielding a stockconcentration of 46 ng/ml (this value was stated on the certificate ofanalysis provided with this lot of kit). 150 ul of the reconstitutedstandard was diluted with 540 ul standard diluent to produce the firstpoint in the standard curve, 10,000 pg/ml. The remaining points werethen prepared using 1:2 dilutions in sample buffer.

It was observed that dilution has a significant effect on the clinicalseparation between PJI positive and negative samples (FIG. 13). Dilutionof 1:1000 seems insufficient, and for higher dilutions there aretradeoffs in separation, especially for challenging high-valuenegatives. The results demonstrate that dilution factors yield anoptimal dilution at which positive and negative separation is maximized.Without wishing to be bound by any particular theory, it is believedthat the desired dilution is between 5,000 and 10,000.

Example 4 HNP1-3 Clinical Study of Synovial Fluid

The commercially available Human HNP1-3 ELISA kit from Hycult Biotechwas used for determining human HNP1-3 concentrations in synovial fluid.Experiments were performed to correlate HNP1-3 levels with the presenceof Periprosthetic Joint Infection (PJI) in synovial fluid synovial fluidfrom human patients. The human HNP1-3 ELISA is a ready-to-use sandwichtype, solid-phase, enzyme-linked immunosorbant assay. Samples, controlsand standards were incubated in microplate wells coated with antibodiesrecognizing human HNP1-3. Biotinylated tracer antibodies provided withthe kit bound to the captured human HNP1-3. Streptavidin-peroxidaseconjugate then binds to the Biotinylated tracer antibody and reactedwith the TMB substrate. The enzyme reaction was stopped by the additionof oxalic acid. The absorbance at 450 nm was measured by aspectrophotometer. Human HNP1-3 concentrations in synovial fluid sampleswere measured by plotting the absorbance of a standard curve versus thecorresponding concentrations of the human HNP1-3 standards.

The materials and methods employed in the experiments disclosed hereinare now described.

Materials and Methods

Clinical Sample

Based on the pilot studies regarding assessing the compatibility of thecommercial kit with the synovial fluid matrix, a clinical assessment of9 different synovial fluid sample patient cohorts were used for theclinical study (Table 10). 71 total synovial fluid samples from thein-house collection were tested in this study. The signal detectiontheory based on ROC curve was used to graphically represent the fractionof true positive rate vs. the false positive rate and to calculate thebest cut-off value for diagnostic decision making.

TABLE 10 Sample Cohort Descriptions Number of Category samplesDescription Classification Pseudo gout 2 Challenging native group −5Gout 3 Challenging native group −4 Mom 1 Challenging Joint group −3 OA15 Challenging native group −2 Aseptic 22 Negative cohort −1Infected-culture 20 Positive cohort 1 positive Infected-culture 3Positive cohort 2 negative Equivocal 3 Positive samples that 3 infectedlack sufficient results to make an unambiguous classification asinfected. For information only and not included in final analysisNative-infected 1 Non-joint positive group 4 Total 70

Storage Conditions Specified in Kit Manual

Upon receipt, individual components were stored at 2-8° C. (withoutfreezing. Components beyond the expiration date printed on the kit labelwere not used. The standard, tracer and streptavidin-peroxidase werestable in lyophilized form until the expiration date indicated on thekit label, if stored at 2-8° C. The exact concentration of the standardwas indicated on the label of the vial and the Certificate of Analysis.Once reconstituted, tracer and streptavidin-peroxidase were stable for 1month if stored at 2-8° C. Once reconstituted, streptavidin-peroxidasecan have a white blurred appearance. Once reconstituted, standard isstable for 1 month if stored at −20° C. Upon receipt, foil pouch aroundthe plate was vacuum-sealed and un-punctured. Any unused strips wereimmediately returned to the foil pouch containing the desiccant pack andresealed along the entire edge of the zip-seal. Quality guaranteed for 1month if stored at 2-8° C.

Sample Preparation

Synovial fluid samples were processed by centrifugation at 2,200 RPM for10 minutes to remove cell debris and other contaminants. Samples arealiquoted and stored at −80° C. until used in assay. Samples were thawedto room temperature and mixed gently before assay was initiated. Sampleswere diluted in 1.5 ml polypropylene tubes using the dilution buffersupplied by Hycult kit HK317-02. The specified dilution was accomplishedfor each sample using a vortex to ensure dispersion of synovial fluid.Samples were not diluted according to product insert. Diluted sampleswere transferred to a round bottom polypropylene 96-well microplate inthe order of the experimental plate layout.

Reagent Preparation

All reagents were equilibrated to room temperature prior to use. Washbuffer was prepared by mixing 20 ml of 40× wash buffer supplied with theHK317-02 kit and 780 ml of diH2O. Dilution buffer was prepared by mixing20 ml of 10× dilution buffer with 180 ml of diH2O. No crystals wereobserved in the concentrated dilution buffer. Standard dilution bufferwas prepared by mixing 10 ml of the 10× plasma diluent with 90 ml of theprepared dilution buffer. Standard solution was prepared byreconstituting lyophilized standard with 0.5 ml of diH20 to get a 46ng/ml stock concentration. A standard curve was prepared inpolypropylene tubes by a serial dilution of the reconstituted standardsolution.

Tracer solution was prepared by reconstituting lyophilized Biotinylatedtracer antibody with 1 ml of diH20. One part reconstituted tracer wasdiluted with 11 parts of dilution buffer.

Streptavidin-peroxidase solution was prepared by reconstituting thelyophilized conjugate with 1 ml of diH2O. Required volume of conjugatesolution was prepared by diluting 1 part reconstituted conjugate with 23parts of dilution buffer.

Standard Curve Preparation

Standard solution was prepared by reconstituting lyophilized standardwith 0.5 ml of diH20 to get a 46 ng/ml stock concentration. Furtherdilutions were made using standard dilution buffer. A standard curve wasprepared in polypropylene tubes using a 1:2 serial dilution of thereconstituted standard. The first standard dilution of the reconstitutedstandard was calculated to achieve a 10,000 pg/ml concentration.

Pilot Study and Clinical Study ELISA Protocol

100 ul in duplicate of standard, samples, and controls were transferredinto appropriate wells, preferably without touching the side or bottomof the wells. The tray was covered and air bubbles were eliminated bytapping the tray without splashing the liquid onto the cover. The stripsor plate were incubated for 1 hour at room temperature. Then the plateswere washed with wash buffer. After washing, 100 ul of diluted tracerwas added to each well with an incubation time of 1 hour at roomtemperature. The plates are then washed to remove the tracer solution.After washing, 100 ul of diluted streptavidin-peroxidase was added toeach well with an incubation time of 1 hour at room temperature. Afterthe incubation time, streptavidin-peroxidase was removed by washing theplates following by the addition of 100 ul of TMB substrate to each wellwith an incubation time of 15 minutes at room temperature (avoidingexposing the micro well strips to direct sunlight). The reaction wasstopped by adding 100 ul of stop solution and the plate was read within30 minutes after addition of stop solution at 450 nm using a platereader, following the instructions provided by the instrument'smanufacturer.

The results of the experiments presented in this Example are nowdescribed.

Human neutrophil alpha-defensins (also called Human Neutrophil Peptides,HNP) belong to the family of cationic trisulfide-containing microbicidalpeptides. There are three highly homologous human defensins stored inazurophilic granules of polymorphonuclear leukocytes, HNP 1-3. Theyaccount for about 5% of total PMN protein and comprise about 99% of thetotal defensin content of the neutrophils with traces of HNP-4. HNP-1,HNP-2 and HNP-3 are encoded by two genes DEFA1 and DEFA3 localized tochromosome 8. DEFA1 and DEFA3 encode identical peptides except theconversion of the first amino acid from alanine in HNP-1 to asparticacid in HNP-3; HNP-2 represent N-terminally truncated iso-form lackingthe first amino acid.

Activation of neutrophils leads to rapid release of HNP. HNP can bemeasured in plasma during infection and inflammation. Micromolarconcentrations of HNP are described in septic blood, while in normalplasma very low levels of HNP are present. The results presented hereincorrelate high HNP levels with septic synovial fluid. HNP1-3 was foundto be a desired biomarker for use in diagnosing periprosthetic jointinfection. The ROC analysis of the data derived a cut-off at 7720 ng/ml.The results demonstrate an AUC of 1.0 with 100% specificity and 100%sensitivity with prosthetic joint samples (Table 11). FIG. 14demonstrates a clinical plot showing that HNP1-3 can be a biomarker forperiprosthetic joint infection in synovial fluid from joint samples.FIG. 15 shows the results from samples tested including synovial fluidfrom native joints.

TABLE 11 Assay performance data Cut-off (derived by ROC analysis Spe-Sen- Spe- Sen- on joint cificity sitivity cificity sitivity BiomarkerAUC samples) Joint Joint ALL ALL HNP1-3 1.000 7720 ng/mL 100.0 100.097.67 100.0

Example 5 BPI Clinical Study of Synovial Fluid

The commercially available Human BPI ELISA kit from Hycult Biotech wasused for determining human BPI concentrations in synovial fluid.Experiments were performed to correlate BPI levels with the presence ofPeriprosthetic Joint Infection (PJI) in synovial fluid synovial fluidfrom human patients. The human BPI ELISA is a ready-to-use sandwichtype, solid-phase, enzyme-linked immunosorbant assay. Samples, controlsand standards were incubated in microplate wells coated with antibodiesrecognizing human BPI. Biotinylated tracer antibodies provided with thekit bound to the captured human BPI. Streptavidin-peroxidase conjugatebound to the Biotinylated tracer antibody and was able to react with theTMB substrate. The enzyme reaction was stopped by the addition of oxalicacid. The absorbance at 450 nm was measured by a spectrophotometer.Human BPI concentrations in synovial fluid samples were measured byplotting the absorbance of a standard curve versus the correspondingconcentrations of the human BPI standards.

The materials and methods employed in the experiments disclosed hereinare now described.

Materials and Methods

Clinical Sample

Based on the pilot studies regarding assessing the compatibility of thecommercial kit with the synovial fluid matrix, a clinical assessment of9 different synovial fluid sample patient cohorts were used for theclinical study (Table 10). 71 total synovial fluid samples from thein-house collection were tested in this study. The signal detectiontheory based on ROC curve was used to graphically represent the fractionof true positive rate vs. the false positive rate and to calculate thebest cut-off value for diagnostic decision making.

111 different synovial fluid samples from the in-house synovial fluidcollection were evaluated. This study was designed to run ELISAs forHNP1-3 and Ela2 alongside BPI in order to expand on the HNP1-3 clinicalstudy discussed elsewhere herein with more samples. There were 51classified synovial fluid samples tested in this experiment that werealso tested in a previous clinical study in order to compare ROCanalysis of biomarkers. The signal detection theory based on ROC curvewas used to graphically represent the fraction of true positive rate vs.the false positive rate and to calculate the best cut-off value fordiagnostic decision making.

TABLE 12 Sample Cohort Descriptions Number of Category samplesDescription Classification Unclassified 55 Insufficient data accompanied−7 with sample to classify Pseudo gout 3 Challenging native group −5Gout 0 Challenging native group −4 Mom 1 Challenging Joint group −3 OA 3Challenging native group −2 Aseptic 24 Negative cohort −1Infected-culture 15 Positive cohort 1 positive Infected-culture 1Positive cohort 2 negative Equivocal 8 Positive samples that lack 3infected sufficient results to make an unambiguous classification asinfected. For information only and not included in final analysisNative-infected 1 Non-joint positive group 4 Total 111

Reagent Preparation

All reagents were equilibrated to room temperature prior to use.Wash/dilution buffer B was prepared by mixing 20 ml of the 40×wash/dilution buffer A supplied with the HK314-02 kit with 380 ml ofdiH2O. Alternatively, Wash/Dilution buffer B was prepared by mixing 40ml of 20× wash/dilution buffer B with 360 ml of diH2O. Finalwash/dilution buffer was prepared by mixing buffer A and buffer Btogether.

Standard solution was prepared by reconstituting lyophilized standardwith 0.5 ml of diH20 to get a 219 ng/ml stock concentration. A standardcurve was prepared in polypropylene tubes by a serial dilution of thereconstituted standard solution.

Tracer solution was prepared by reconstituting lyophilized Biotinylatedtracer antibody with 1 ml of diH20. 1 part reconstituted tracer wasdiluted with 11 parts of dilution buffer.

Streptavidin-peroxidase solution was prepared by reconstituting thelyophilized conjugate with 1 ml of diH2O. Required volume of conjugatesolution was prepared by diluting 1 part reconstituted conjugate with 23parts of dilution buffer.

Standard Curve Preparation

Standard solution was prepared by reconstituting lyophilized standardwith 0.5 ml of diH20 to get a 219 ng/ml stock concentration. Furtherdilutions were made using wash/dilution buffer.

A standard curve was prepared in polypropylene tubes using a 1:2.5serial dilution of the reconstituted standard. The first standarddilution of the reconstituted standard was calculated to achieve a25,000 pg/ml concentration.

The results of the experiments presented in this Example are nowdescribed.

The antimicrobial protein BPI (Bacterial Permeability Increasingprotein) is a 55 kDa protein found in the primary azurophilic granulesof human neutrophils and has also been detected on the surface ofneutrophils, small intestinal and oral epithelial cells. BPI is abactericidal compound that is present in polymorphonuclear cells and inlower levels in the specific granules of eosinophils. BPI possesses highaffinity toward the lipid A region of lipopolysaccharides (LPS) thatcomprise the outer leaflet of the gram-negative bacterial outermembrane. Binding of BPI to the lipid A moiety of LPS exerts multipleanti-infective activities against gram-negative bacteria: 1)cytotoxicity via sequential damage to bacterial outer and inner lipidmembranes, 2) neutralization of gram-negative bacterial LPS, 3)opsonization of bacteria to enhance phagocytosis by neutrophils. Airwayepithelial cells constitutively express the BPI gene and produce the BPIprotein and, therefore, BPI may be a critical determinant in thedevelopment of LPS-triggered airway disease. Inflammation induced by LPSpossibly contributes to the development of rapid airflow decline, aserious and often fatal complication of hematopoietic celltransplantation. In plasma of healthy individuals BPI is present atlevels of <0.5 ng/ml, which increases approximately 10-fold during acutephase responses.

The results presented herein demonstrate elevated BPI concentrations ininfected synovial fluid samples. For the ROC analysis, a backgroundsubtracted signal was used to derive a cut-off of 2.18 O.D. The analysisshowed an AUC of 1.0 with 100% specificity and 100% sensitivity withprosthetic joint samples (Table 13). FIGS. 16 and 17 demonstrateclinical plots showing that BPI is a biomarker for periprosthetic jointinfection in synovial fluid from joint samples.

TABLE 13 Assay performance data Cut-off (derived by ROC analysis usingbackground Specificity Sensitivity Biomarker AUC subtracted data) JointJoint BPI 1.0 2.18 O.D. 100 100

Example 6 Sepsis Panel Clinical Screen

The following experiments were performed to identify potentialbiomarkers for periprosthetic joint infection. The entire MilliporeSepsis Panel III was used to screen an expanded cohort of samples toestablish a clinical data set that can be used for ROC and other cutoffanalysis.

Briefly, Millipore Sepsis Panel 3 (HSP3MAG-63k) was used to source allmaterials for this experiment. After allowing the kit contents to reachroom temperature, the magnetic beads were prepared. Each individual beadvial was sonicated for 30 seconds, than vortexed for 1 minute. 150 ul ofeach bead solution was combined in the bead mixing vial, and the mixturewas brought to a total volume of 3.0 ml using the bead diluent.

The standard was reconstituted with 250 ul of diH2O and allowed to sitfor 15 minutes at room temperature. Standards were then prepared using a6-step 1:4 serial dilution, using the assay buffer as a diluent.

Selected synovial fluid samples were then diluted 1:1,000 in assaybuffer. 50 ul of assay buffer was added to each well on the assay plate,and then 25 ul/well of sample or standard was added as appropriate. 25ul/well of thoroughly vortexed bead mixture was then added. The platewas sealed and incubated for 2 hours at 25* at shake setting 5 on theJitterbug plate shaker.

The plate was washed using the standard Luminex wash protocol (DPBS w/0.05% Tween as wash buffer, Biotek automated washer for all steps: 1minute incubation on magnet, aspiration and dispense of wash buffer, 30seconds shaking on the jitterbug, repeated 3×). 25 ul/well of detectionantibody cocktail was then added to the plate. After one hour ofincubation at 25* at shake setting 5 on the Jitterbug plate shaker, 25ul/well of Strep-PE detection molecule was added to each well. The platewas incubated for 30 min at 25* at shake setting 5 on the Jitterbugplate shaker.

The full wash protocol was run, then 150 ul/well of Millipore 1× washbuffer was added. The plate was agitated for 10 minutes to fullyresuspend beads, then read on the LX200 unit utilizing BioPlex software.

The results demonstrate that Elastase 2, Resistin, NGAL, thrombospondin,and Lactoferrin are biomarkers for periprosthetic joint infection.

Example 7 Biomarkers for Joint Infection

Using an established sample bank of synovial fluid that included someproblematic samples, experiments were designed to further evaluateadditional biomarkers. The series of markers include IL-1β, IL-6, IL-8,TNFα, G-CSF, IL-1a, VEGF, IP-10, BFGF (aka FGF2), CRP, a2M, SKALP, HNEEnzyme assay, LE Strip, Lactoferrin, Lipocalin-2/NGAL, NeutrophilElastase-2 (ELA2), Resistin, Thrombospondin-1 (TSP-1), HNP1-3, and BPI.Each marker was evaluated with a limited set of positive and negativesamples and evaluated with a larger cohort (Table 10) if there was goodseparation between the samples. The overall assay performance is listedin Table 14.

TABLE 14 ROC Analysis Cut-off (derived by Cut-off ROC analysis Range onjoint (same unit Specificity Sensitivity Specificity SensitivityBiomarker AUC samples) as cutoff) Joint Joint ALL ALL HNP1-3 1.000 7720ng/mL  3334-10946 100.0 100.0 97.67 100.0 ELA-2 1.000 942 ng/mL 721-19000 100.0 100.0 97.67 100.0 NGAL 0.998 1,644 ng/mL 1100-320095.65 100.0 93.02 100.0 Resistin 0.996 82.9 ng/mL  53-112 95.65 100.093.02 100.0 Thrombospondin 0.996 136 ng/mL 131-141 95.65 100.0 92.86100.0 Lactoferrin 0.996 2,993 ng/mL 1200-4700 95.65 100.0 93.02 100.0IL-1β (MSD) 0.991 33.25 pg/mL 30-35 95.65 100.0 97.67 100.0 IL-8 (MSD)0.989 6794 pg/ml 6725-6860 95.65 100.0 97.67 100.0 CRP 0.975 11412 ng/mL11000-12000 95.65 91.30 93.02 91.67 TNFα (MSD) 0.960 66.42 pg/mL 65-88100.0 82.61 IL-6 (MSD) 0.940 3472 pg/mL 1965-5000 95.65 91.30 HNE 0.926552.8 ng/mL 521-584 100.0 95.45 97.67 95.65 a2M 0.855 73.45 pg/mL 70-7678.26 77.27 72.09 78.3 VEGF 0.851 2565 pg/mL 2500-3300 82.61 78.26 FGF20.785 2.25 pg/mL  1-12 95.65 65.22 G-CSF 0.712 94.35 pg/mL  74-100 73.9173.91 SKALP 0.675 3721 pg/mL 2100-3800 84.21 52.94 IP-10 0.582 5003pg/mL 4500-5800 78.26 56.52 LBP Orsomucoid

Example 8 Detection of Hyaluronic Acid in Synovial Fluid

Hyaluronic acid (HA) is a large carbohydrate polymer that is a majorcomponent of synovial fluid. HA increases the viscosity of the synovialfluid and thereby increases its quality as a lubricant. While HA isfound in a variety of tissues (including connective, epithelial, andneural), the concentration of hyaluronic acid is greatly increased insynovial fluid relative to other tissues, including serum. As such, thequantitation of HA in biological samples could serve as a means todistinguish synovial fluid from contaminating serum.

A series of human synovial fluid (from an internal collection) and serumsamples (purchased from Bioreclamation) were probed for the presence ofHA using a Quantikine ELISA kit produced by R&D Systems (cat# DHYAL0).

The results presented herein demonstrate that when diluted 1:10 or1:100, synovial fluid samples produce an OD signal that was well abovethe signal generated by the 40 ng/mL standard, the highest standardrecommended by the R&D protocol. As these levels were at the maximumabsorbance at 450 nm, synovial fluid sample could not be differentiatedform one another for the concentration of HA, unlike the serum samples,where some variation could be observed. Nonetheless, there was a clearseparation in the signal generated by serum or synovial fluid sample(FIG. 18). FIG. 18 depicts a representative standard curve from (left)and a comparison of OD450 signal for synovial and serum samples(right;-1 indicated a serum sample, 1a synovial fluid sample).

Next, synovial fluid samples were diluted well beyond 1:100. Even at1:100,000, raw signal for synovial samples were detectable abovebackground, though a 1:50,000 dilution was adequate to bring the samplesinto range. Synovial fluid samples were found to have HA concentrationsranging from 0.529 ug/ml to 2.575 ug/ml. Even at a 1:1,000 dilution, HAlevels in serum were not detectable. For both a “high” and “low” SFsample, dilution with serum produces equal and expected changes in theconcentration of HA.

The results presented herein demonstrate that HA is a valid marker fordistinguishing synovial fluid from serum. In some instances, thesensitivity of this assay prefers that synovial fluid be diluted1:50,000 any yet still allows for very clear detection of whether or notany synovial fluid is present in a sample.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1. (canceled)
 2. A system for diagnosing joint infection in a subject,wherein the system comprises: a) a first region comprising a firstdetection reagent that detects the presence of the biomarker for jointinfection in synovial fluid, wherein the first detector reagentspecifically binds the biomarker, b) a second region comprising aninternal control detector reagent comprising an immunoglobulin; whereindetection of the biomarker diagnoses joint infection in the subject withat least 90% accuracy.
 3. (canceled)
 4. The system of claim 2, whereinthe biomarker is selected from the group consisting of defensin, HNP1-3,ELA-2, BPI, NGAL, Resistin, Thrombospondin, Lactoferrin, IL-1β, IL-8,CRP, TNFα, IL-6, HNE, a2M, VEGF, FGF2, SKALP, IP-10, LMP, Orsomucoid,and any combination thereof.
 5. (canceled)
 6. The system of claim 2,wherein the system has a sensitivity and specificity of at least 90% forjoint infection.
 7. The system of claim 2, wherein the joint is selectedfrom the group consisting of a native joint and a replacement joint. 8.(canceled)
 9. A method of diagnosing joint infection in a subjectcomprising detecting the presence of a biomarker in synovial fluidobtained from a joint in the subject, the method comprising applyingsynovial fluid obtained from a joint in the subject to a system, whereinthe system comprises: a) a first region comprising a first detectionreagent that detects the presence of the biomarker for joint infectionin synovial fluid, wherein the first detector reagent specifically bindsthe biomarker; b) a second region comprising an internal controldetector reagent comprising an immunoglobulin; wherein detection of thebiomarker diagnoses joint infection in the subject with at least 90%accuracy.
 10. The method of claim 9 comprising: a) contacting thesynovial fluid obtained from the joint in the subject with an assaybuffer; b) applying the synovial fluid so contacted to the system. 11.(canceled)
 12. The method of claim 9, wherein the biomarker is selectedfrom the group consisting of defensin, HNP1-3, ELA-2, BPI, NGAL,Resistin, Thrombospondin, Lactoferrin, IL-1β, IL-8, CRP, TNFα, IL-6,HNE, a2M, VEGF, FGF2, SKALP, IP-10, LMP, Orsomucoid, and any combinationthereof.
 13. (canceled)
 14. The method of claim 9, wherein the systemhas a sensitivity and specificity of at least 90% for joint infection.15. The system of claim 9, wherein the joint is selected from the groupconsisting of a native joint and a replacement joint.
 16. (canceled) 17.The method of claim 10, wherein the assay buffer dilutes the synovialfluid to enhance the ability to pipette and transfer the synovial fluid.18. The method of claim 10, wherein the assay buffer comprises an agentthat lyses cellular components present in the synovial fluid.
 19. Themethod of claim 10, wherein the agent is a non-ionic surfactant.
 20. Themethod of claim 10, wherein the assay buffer comprises an agent thatpreserves the synovial fluid and stabilizes biomarkers present in thesynovial fluid.
 21. The method of claim 10, wherein the assay buffercomprises an agent that inhibits an interfering component present in thesynovial fluid.
 22. The method of claim 10, wherein the assay buffermaintains a pH in the range of about 6-8.